Techniques in Neuropharmacology – Multiple Neurobiological Techniques for Assessing the CNS
How would you feel with an electrode implanted deep within your brain that delivered mild electrical pulses to change your neural activity? If you happen to be one of the thousands of people suffering from chronic disabling disorders like Parkinson’s disease, chronic pain, or epilepsy, the answer is that you might feel dramatic relief.
Deep brain stimulation therapy is one of the newest techniques to treat debilitating neurological disorders in patients who fail to respond to currently available medications. The treatment involves applying minute amounts of electrical current to precise brain sites to modify the brain signals that cause undesirable symptoms. It involves surgically implanting a fine wire deep into the individual’s brain that is connected by an extension wire placed under the skin to a pacemaker-type electrical generator. The battery-powered generator is surgically placed under the skin near the collarbone and can be programmed by the neurophysiologist to deliver the precise stimulation needed by an individual patient for greatest relief and fewest side effects. Patients can also control the stimulation delivered by using a magnet to increase or decrease the “dosage” as required. This method, recently tested at medical facilities all around the world, demonstrates the creative and practical application of years of basic animal research into neurobiological methods.
Multiple Neurobiological Techniques for Assessing the CNS
The discovery of chemical transmission of information between nerve cells paved the way for the birth of neuropharmacology. Since then, there has been an explosion of research directed toward understanding the nature of brain function and the biology of what makes us human. With the variety and power of new analytical tools and techniques we can look inside the brain to find answers to questions that touch individual lives. Even nonscientists can appreciate the advances in neuroscience research that bring us ever closer to understanding the essence of human behavior aş well as some of the most troubling problems of mankind: dementia, depression, autism, and neurodegenerative disorders.
The new tools provide the means to explore the brain to answer our questions, but it takes disciplined and creative scientific minds and teamwork to pose the right questions and use available tools optimally. The scientific method, utilizing rigorous hypothesis testing under controlled conditions, is the only real method we have to investigate how molecules responsible for nerve cell activity relate to complex human behaviors and thinking. Analysis spans the entire range from molecular genetics to cell function to integrated systems of neuronal networks and finally to observable behavior. To understand the brain requires a convergence of efforts from multiple disciplines that together form the basis of neuroscience: psychology, biochemistry, neuropharmacology, neuroanatomy, endocrinology, computer science, neuropsychology, and molecular biology. Ultimately, the knowledge we acquire depends on integrating information derived from a wide variety of research techniques from all of these fields.
As you might expect, the list of techniques is very long and increases every day. this posts focuses on a few of the more common methods and helps you to understand each method’s purpose as well as some of its potential weaknesses. Perhaps the most important goal of this post is to encourage you, when you read scientific papers, to critically evaluate the methods and the controls used, because the conclusions we draw from experiments are only as good as the methodology used to collect the data.
The essence of neuropsychopharmacology is the use of drugs as a means to modify synaptic activity and subsequent behavior. a future post describes some of the chemical agents used to alter the synthesis, packaging, and release of neurotransmitters as well as prolong or shorten neurotransmitter action by altering metabolism or reuptake. Most important is the use of agonists to mimic and antagonists to reduce normal neurotransmitter action at the receptor.
Because synaptic activity is so important, the first part of this post emphasizes techniques that look at the location and function of neurotransmitters and neurotransmitter receptors. The methods are both in vivo, meaning observed in the living organism, and in vitro, which is measurement outside the living body (traditionally in a test tube). We also look at a variety of rather remarkable imaging techniques that permit us to visualize the activity of the living human brain. Since genetic engineering is an increasingly powerful tool, we will describe its use in neuropharmacology. The second part of the post focuses on behavioral pharmacology. Behavior, mood, and thinking represent the focus of neuropsychopharmacology, so it is of equal importance to understand and critically evaluate the techniques used to quantify behavioral changes. Both the biochemical and the behavioral techniques selected will be used in subsequent posts. Feel free to return to this posts to review a method when you encounter it later.
Stereotaxic surgery is needed for accurate in vivo measures of brain function
The classic techniques of physiological psychology (lesioning, microinjection, and electrical recording) are equally important in understanding the action of psychoactive drugs. Stereotaxic surgery is an essential technique in neuroscience that permits a researcher to implant one of several devices into the brain of an anesthetized animal with significant precision. The stereotaxic device itself is essentially a means to stabilize the animal’s head in a fixed orientation so that the carrier portion can be moved precisely in three dimensions to place the tip of an electrode or drug delivery tube in a predetermined brain site. The brain site coordinates are calculated using a brain atlas, which is a collection of frontal sections of brain of the appropriate species in which distances are measured from skull surface features. Accuracy of placement is determined histologically after the experiment is complete. The halo bracket is the equivalent apparatus used in human neurosurgery, and the target site is identified with a computerized imaging technique like magnetic resonance imaging (MRI) or computerized tomography (CT).
Lesioning and microinjection Experimental ablation, or lesioning, uses a stereotaxic device to position a delicate electrode, insulated along its length except for the exposed tip, deep within the brain. The tissue at the tip is destroyed when a very-high-frequency radio current is passed through the electrode to heat the cells. The rationale of the experiment is that a comparison of the animal’s behavior before and after the lesion will tell us something about the function of that brain area.
Electrolytic lesions destroy all tissue at the tip of the electrode, including cell bodies, dendrites, and axons. Alternatively, a neurotoxin (a chemical damaging to nerve cells) can be injected via a cannula (a hollow tube inserted like an electrode) to destroy cells. Of course, the same type of cannula can be used to administer drugs or neurotransmitters that stimulate cells in the central nervous system (CNS) before evaluating behavior. Chemical lesions have the advantage of being significantly more specific because neurotoxic chemicals, such as kainic acid or ibotenic acid, kill the cell bodies in the vicinity of the cannula tip but spare the axons passing through the same area. In either case, this procedure can be used to identify the brain area responsible for a drug-induced change in behavior. For instance, we might wonder which brain area is responsible for the reinforcing effects of a drug like amphetamine. Suppose that after lesioning the nucleus accumbens in the diencephalon, we find that rats no longer will self-administer amphetamine by pressing a lever in an operant chamber (see the section on operant behavior later in the post). We may want to conclude that the nucleus accumbens is responsible for reinforcement, but lesion studies must always be evaluated cautiously. Even when a lesion changes behavior, we still don’t know what specific function that brain area served. In our example, further investigation would be needed to determine how the lesion interfered with the self-administration. Does the nucleus accumbens modulate reinforcement? Or is it possible that the animal lost motor control or failed to remember the appropriate response? Furthermore, because of the small size of brain structures and their overlapping nature, the possibility exists that behavioral change is due to damage to adjacent brain regions.
The lesioning technique has always been a valuable tool to examine the relationship between brain structure and function in animals. In humans, of course, lesions cannot be produced intentionally, but accidents, trauma to the brain, strokes, and tumors (“accidents of nature”) all provide a means to investigate the relationship between brain damage and function. Psychology students will certainly remember the story of Phineas Gage, whose skull and brain were penetrated by a long steel rod in a blasting accident. His case history has become famous for being an example of profound behavioral changes following traumatic brain injury. Previously a mild-mannered man and competent foreman of a work crew, after the accident Gage demonstrated childish behavior and an inability to organize his daily activities, displaying frequent uncontrolled outbursts and episodes of violence. However, several significant problems exist in evaluating such case studies. First, although behavioral measures and neuropsychological testing after injury can identify deficits in function, it is rare that skills were evaluated prior to the injury. For this reason it is difficult to know to what extent the functioning changed as a result of the injury. Second, until very recently, there was no way to know specifically where the brain damage occurred. The development of scanning techniques like CT and MRI have greatly improved the ability to identify quite specifically the locus of damage. Third, “accidents of nature” produce unique damage to brain structures in each individual, so generalizations to a larger population are unwarranted.
Because neuropharmacology is interested in neurochemical regulation of behavior, the lesioning techniques used are often specific for a neural pathway utilizing a particular neurotransmitter. These specific neurotoxins are most often injected directly into the brain, where they are taken up by the neurons’ normal reuptake mechanism. Once inside the cell, the toxin destroys the cell terminal. In this way, behavioral measures made before and after a neurotoxic lesion tell us about the role of the neurotransmitter in a particular behavior. For example, intracerebroventricular administration of 6-hydroxydopamine produces nerve terminal degeneration in both noradrenergic and dopaminergic cells and profound neurotransmitter depletion. More selective effects are achieved when the neurotoxin is injected directly into a target area. Earlier we suggested that lesioning the nucleus accumbens reduced self-administration of amphetamine in rats. We might further test our understanding of the role of the nucleus accumbens in reinforcement by selectively destroying the large number of dopamine cell terminals in that area using the neurotoxin 6-hydroxydopamine before evaluating the drug-taking behavior.
Microdialysis A different technique that uses stereotaxic surgery is microdialysis. Although researchers have been able to measure neurotransmitters released from brain slices in vitro for many years, microdialysis lets us measure neurotransmitters released in a specific brain region while the subject is actively engaged in behavior.
The technique requires a specialized cannula made of fine, flexible tubing that is implanted-stereotaxically. The cannula is sealed along its length except at the tip, allowing investigators to collect material in extracellular fluid at nerve terminals at precise sites even deep within the brain. Artificial cerebrospinal fluid (CSF) is gently moved into the microdialysis cannula by a pump. The CSF in the cannula and in the extracellular fluid are identical except for the material to be collected. Based on the difference in concentration, the chemicals of interest move across the membrane from the synaptic space into the cannula. A second pump removes the CSF from the cannula into a series of tubes, to be analyzed by high-performance liquid chromatography (HPLC) or another method.
A major improvement over older collection methods is that only tiny amounts of material need to be collected for accurate measurement. The improved accuracy is due to the development of highly sensitive analytic techniques (such as HPLC), which can be combined with microdialysis collection.
HPLC, like other types of chromatography, serves two purposes. First, chromatography separates the sample into component parts depending on characteristics of the sample, such as molecular size or ionic charge. Second, the concentration of the molecules of interest can be determined.
Microdialysis is important to neuropsychopharmacology because it can be used in several types of experiments combining biochemical and behavioral analyses. For example, we might evaluate the released neurochemicals during ongoing behaviors such as sleep and waking, feeding, or operant tasks to provide a window into the functioning CNS. Second, we might investigate the effects of drugs on extracellular concentrations of neurotransmitters in selected brain areas. Since the sample collection can be made in freely moving animals, correlated changes in behavior can be monitored simultaneously. Finally, the collection of extracellular materials at nerve terminals following discrete electrical or chemical stimulation of neural pathways is another valuable role.
A second method used to measure neurotransmitter release is in vivo voltammetry. Whereas microdialysis collects samples of extracellular fluid for subsequent analysis, in vivo voltammetry uses stereotaxically implanted microelectrodes to measure neurochemicals in the extracellular fluid of freely moving animals. In voltammetry a very fine electrode is implanted and a small electrical potential is applied. Changes in the current flow at the electrode tip reflect changes in the concentration of electroactive substances such as neurotransmitters or their metabolites. A major advantage is that because the measurements are made continuously and require as little as 15 milliseconds to complete, researchers can evaluate neurotransmitter release as it is occurring in real time.
Electrophysiological stimulation and recording In a similar fashion, implanted macroelectrodes can be used to activate cells at the tip while evaluating the change in animal behavior during stimulation. The minute amount of electric current applied changes the membrane potential of those cells and generates action potentials. The action potentials in turn cause the release of neurotransmitter at the cell terminals to mimic normal synaptic transmission. Hence the electrical stimulation should produce biobehavioral effects that are similar to those seen upon injection of the natural neurotransmitter or neurotransmitter agonists into the brain. In addition, one would expect that stimulation of a given cell group should produce effects opposite those of a lesion at the same site. Macroelectrodes can also be used to record the summated electrical response of thousands of neurons in a specific brain region following drug treatment or other experimental manipulation in a freely moving animal. If we had found, for example, that lesioning the periaqueductal gray (PAG) in the midbrain prevented the pain-reducing effects of morphine, we might want to find out what effect activating those PAG neurons has. What we would find is that if electrodes implanted in the PAG are activated, the animal fails to respond to painful stimuli. Likewise, if pain-killing opioids like morphine or codeine are microinjected into that brain area via an indwelling cannula, the animal also demonstrates profound analgesia.
In the opening paragraphs of this post, you read about patients who have benefited from the many years of animal research into the stereotaxic implantation of electrodes into the brain and electrical stimulation. The adaptation of the technique to humans being treated for Parkinson’s disease. Small pulses of electric current applied to the thalamus cause the cells to fire and release neurotransmitter, which reduces the tremor on the opposite side of the body.
An alternative to macroelectrode recording, which is a summation of electrical activity in a brain region, is singleunit recording, which uses microelectrodes. Stereotaxically implanting a fine-tipped electrode either into a single cell (intracellular recording) or into the extracellular fluid near a single cell (extracellular recording) monitors the response of individual cells under various conditions. Intracellular recording must utilize an anesthetized animal, because the electrode must remain in a precise position in order to record the membrane potential of the cell. An advantage of extracellular single-cell recording is that it can be done in a mobile animal. The downside to extracellular recording is that the electrode records only the occurrence of action potentials in the nearby neuron and cannot monitor the change in the cell’s membrane potential. Returning to our earlier example of morphine action, we find that the drug produces strong selective inhibition of neurons in the spinal cord, which prevents the projection of pain information to higher brain centers, thereby contributing to the analgesic effect.
In addition to measuring membrane potentials of groups of cells and single cells, thanks to the Nobel Prize-winning research of Neher and Sakmann conducted in the 1970s neuroscientists can also study the function of individual ion channels, which collectively are responsible for the membrane potential. The technique, known as patch clamp electrophysiology, works best with individual cells in culture but can also be used on exposed cells in slices of brain. The method involves attaching a recording micropipette to a piece of cell membrane by suction. When the pipette is pulled away, a small membrane patch containing one or more ion channels remains attached. The subsequent electrical recording through the pipette represents in real time the channel opening, the flow of ions (electrical current) during the brief period when it is open, and the channel closing.
Neurotransmitters, receptors, and other proteins can be quantified and visually located in the CNS
To both quantify and locate neurotransmitters and receptors in the CNS, several methods are required. To count or measure a particular molecule, a “soup” method is often used, in which a tissue sample is precisely dissected out and ground up, creating a homogenate before being evaluated. Homogenates are used in any one of many possible neurochemical analyses which are referred to as assays. In contrast, for localization, the landmarks of the tissue and relationship of structures must be preserved, so the visualization method is done on an intact piece or slice of tissue. Hence, when we want to measure the number of receptors in a particular brain area we are likely to use a radioligand binding assay in a tissue homogenate, but if we want to see where in the brain particular receptors are located (as well as measure them) we are more likely to use a slice preparation with autoradiography. Table 1 summarizes the “soup” and “slice” techniques described in the following section of the post.
TABLE 1 Methods Used to Quantify and Visualize Target Molecules in the Nervous System
|Target molecule||Tissue extract assay to quantify||Brain slice preparation to visualize|
Receptors and other proteins mRNA
|Radioligand binding Radioimmunoassay (RIA) Dot blot or Northern blot||Receptor autoradiography Immunocytochemistry(ICC) In situ hybridization (ISH)|
Radioligand binding To study the number of receptors in a given brain region and their affinity for drugs, the radioligand binding method was developed. Once the brain region we are interested in is dissected out,
it is ground up to make a homogenate. A ligand (usually a drug or chemical) that is radioactively labeled (now called the radioligand) is incubated with the tissue under conditions that optimize its binding. After a brief time, any radioligand that has not bound is removed, often by washing and filtering. The amount of radioligand bound to the tissue is then measured with a scintillation (or gamma) counter and reflects the number of receptors in the tissue.
Although the binding procedure is quite simple, interpretation of the results is more complex. How can we be sure that the radioligand is actually binding to the specific biological receptors of interest, rather than to other sites based on artifacts of the procedure? Several criteria that must be met include (1) specificity; (2) saturability; (3) reversibility and high affinity; and (4) biological relevance. Specificity means that the ligand is binding only to the receptor we are concerned with in this tissue and to nothing else. Of course, drugs often bind to several receptor subtypes, but they may also attach to other cell components that produce no biological effects. To measure the amount of a ligand that binds to the site that we are concerned with, we add very high concentrations of a nonradioactive competing ligand to some tubes to show that most of the radioactive binding is displaced. That which remains is likely to be nonspecifically bound to sites such as assay additives (e.g., albumin) or cellular sites (e.g., enzymes) that we are less interested in at the moment. Nonspecific binding is subtracted when the data are calculated for specific binding. When binding to specific subtypes of receptors is necessary, ligands must be designed to distinguish between the receptor proteins.
Saturability means that there are a finite number of receptors in a given amount of tissue. By adding increasing amounts of radioligand to a fixed amount of tissue, one would expect to see gradual increases in binding until all sites are filled. Binding in the assay must also be reversible, because a neurotransmitter in vivo will bind and release many times to initiate repeated activation of the cellular action. This reversibility is demonstrated in binding assays because the radioactive ligand can be displaced by the same drug that is not radiolabeled. The unbinding (dissociation) of the ligand from the receptor must also be consistent with the reversal of physiological effects of the ligand.
Ideally, the binding of chemically similar drugs should correlate with some measurable biochemical or behavioral effect. For example, the classic antipsychotic drugs all bind to a particular subtype of receptor (D2) for the neurotransmitter dopamine. Not only do the drugs in this class bind to the D2 receptor, but their affinity for the receptor correlates with the effectiveness of the drugs in reducing the symptoms of schizophrenia. Unfortunately, experiments of this type rarely produce perfectly correlated results in binding affinity and functional potency because drug effects in the intact organism are dependent on many factors in addition to drug-receptor interaction, for example, absorption and distribution.
Receptor autoradiography Receptor binding is a classic tool in neuropharmacology that tells us about receptor number and affinity for a particular drug in a specific piece of brain tissue. When we want to visualize the distribution of receptors within the brain, we use receptor autoradiography. The process begins with standard radioligand binding as described above except that slide-mounted tissue slices rather than ground-up tissue are used. After the unbound radioactively labeled drug is washed away, the slices are processed by autoradiography. The slides are put into cassettes, a specialized autoradiographic film is placed on top of the slides so that it is in physical contact with the tissue sections, and the cassettes are stored in the dark to allow the radioactive material that is bound to receptors to act on the film. The particles that are constantly emitted from the radioactive material in the tissue expose the film and show not only the amount of radioligand bound but also its location. This method is especially good for studying the effects of brain lesions on receptor binding because each lesioned animal can be evaluated independently by comparing the lesioned and nonlesioned sides of the brain. This method might also give us clues about how various psychoactive drugs produce their behavioral effects. For instance, mapping the binding of cocaine in monkey brain shows a distinct pattern of localization and density in selected brain areas. With a clear understanding of anatomical distribution, we can begin to test specific hypotheses regarding the behavioral consequences of activating these receptors using microinjections of receptor-selective agonists and antagonists.
In vivo receptor binding The same autoradiographic processing can be done on brain slices of an animal that had previously been injected in vivo with a radiolabeled drug. The drug enters the general circulation, diffuses into the brain, and binds to receptors. The animal is then killed and the brain is sliced and processed by autoradiography. The technique shows the researcher where a particular drug or neurotransmitter binds in an intact animal. Unfortunately, results with this technique are more difficult to interpret because of the complexities of bioavailability and distribution, diffusion through the blood-brain barrier, and metabolism of the drug. Nevertheless, its potential is tremendous because in vivo binding can be assessed in living human subjects using positron emission tomography (PET) (see the section on brain imaging) to map the pattern of drug-recep- tor binding and correlate it with clinical effects.
Assays of enzyme activity Enzymes are proteins that act as biological catalysts to speed up reaction rates, but they are not used up in the process. We find many different enzymes in every cell, and each has a role in a relatively specific reaction. The enzymes that are particularly interesting to neuropharmacologists are those involved in the synthesis or metabolism of neurotransmitters, neuromodulators, and second messengers. In addition, neuropharmacologists are interested in identifying the conditions that regulate the rate of activity of the enzyme. For example, acute morphine treatment inhibits adenylyl cyclase activity. Adenylyl cyclase is the enzyme that synthesizes the second messenger cyclic adenosine monophosphate (cAMP). However, chronic exposure to morphine produces a gradual but dramatic up-regulation of the cAMP system, suggesting that the second-messenger system acts to compensate for the acute effect of opioid inhibition. It is perhaps one of the best-studied biochemical models of opioid tolerance and is discussed further in a future post.
Sometimes the mere presence of an enzyme in a cell cluster is important since it can be used to identify those cells that manufacture a specific neurotransmitter. The next section describes the use of antibodies and immunocytochemistry to locate enzymes in the brain.
Antibody production Some of the newest methods for identifying and measuring receptors and other proteins are far more specific and sensitive than ever before because they use an antibody. An antibody is a protein produced by the white blood cells of the immune system to recognize, attack, and destroy a specific foreign substance (the antigen). Researchers use this immune response to create supplies of antibodies that bind to specific proteins (receptors, neuropeptides, or enzymes) they want to locate in the brain. The first step is to create an antibody by injecting the antigen (for example, the neuropeptide hypocretin) into a host animal and at various times taking blood samples to collect antibodies. With the antibody prepared, we are ready to look for the peptide in tissue slices using immunocytochemistry. Antibodies can also be used to quantify very small amounts of material using radioimmunoassays (see below).
For immunocytochemistry (ICC), the brain is first fixed (hardened) using a preservative such as formaldehyde. Tissue slices are then cut and incubated with the antibody in solution. The antibody attaches to the antigen wherever cells are present that contain that antigen. In the final step, the antibody is tagged so that the antigen-containing cells can be visualized. This is usually accomplished either by means of a chemical reaction that creates a colored precipitate within the cells or by using a fluorescent dye that glows when exposed to light of a particular wavelength. The researcher can then examine the tissue slices under a microscope and see which brain areas or neurons contain the antigen. The technique is limited only by the ability to raise antibodies. The visualization of cells that contain the neuropeptide hypocretin in the lateral hypothalamus of a healthy human subject. In patients with the sleep disorder narcolepsy, the number of hypocretin neurons is reduced by about 90% (Thannicakal et al., 2000). These results, along with animal experiments using neurotoxin lesioning and genetic modification, suggest that hypocretin in the hypothalamus may regulate the onset of sleep stages. ICC is similar to autoradiography in principle, but it is far more selective because of the use of the antibody (which recognizes only a very specific protein) and much quicker because it does not require the development time of the autoradiographic film.
Radioimmunoassay Antibodies are also useful in quantifying physiologically important molecules in body fluids such as blood, saliva, or CSF, as well as in tissue extracts. Radioimmunoassay (RIA) is based on competitive binding of an antibody to its antigen (the molecule being measured). The use of antibodies makes the procedure highly specific for the molecule of interest and very sensitive.
RIA involves preparing a standard curve of known antigen concentrations against which unknown samples can be compared. The standard curve is created by first combining a preset amount of antibody with a known concentration of radioactively labeled antigen in all the assay tubes. At this point all the tubes are identical, that is, all the antibody would be reversibly attached to radioactive antigen. However, the experimenter then adds different, known concentrations of unlabeled antigen, which compete with the radioactively labeled which compete with the radioactively labeled antigen. The higher the concentration of unlabeled competitor antigen added, the lower the amount of radioactive antigen bound after the mixture has been incubated. The values are plotted as a standard curve and analyzed using appropriate computer software.
To determine how much of the antigen is in any experimental sample, other test tubes are prepared in just the same way except that the samples containing unknown amounts of antigen are added instead of the known antigen. By measuring the amount of radioactive antigen bound in the sample tubes compared to the standard curve, the amount of antigen in the sample can be calculated.
In situ hybridization In situ hybridization (ISH) makes it possible to locate cells in tissue slices that are manufacturing a particular protein or peptide in much the same manner that ICC identifies cells containing a particular protein. ISH is particularly useful in neuropharmacology for detecting the specific messenger RNA (mRNA) molecules responsible for directing the manufacture of the wide variety of proteins essential to neuron function, such as enzymes, structural proteins, receptors, ion channels, and peptides. Because the method detects cells with a precise RNA sequence, it is exceptionally specific and extremely sensitive. Besides locating cells containing specific mRNA, ISH is also used to study changes in regional mRNA levels after experimental manipulations. The amount of mRNA provides an estimate of the rate of synthesis of the particular protein.
As you recall from an earlier post, the double strands of DNA and corresponding mRNAhave unique base-pair sequences responsible for directing the synthesis of a particular protein with its unique amino acid sequence. ISH depends on the ability to create probes by labeling single-stranded fragments of RNA made up of base-pair sequences complementary to those of the mRNA of interest. After the single strands are prepared, they are labeled radioactively or with dyes. When the tissue slices or cells are exposed to the labeled probe, the probe attaches (binds, or hybridizes) to the complementary base-pair sequences. After incubation, the tissue is washed and dehydrated before being placed in contact with X-ray film or being processed in other ways for visualization of cells containing the specific mRNA. The technique is extremely sensitive and can detect a very small number of cells that express a particular gene. If the researcher is interested only in measuring the amount of mRNA rather than visualizing its location, hybridization can be done using a tissue homogenate rather than a tissue slice. Two available methods of ISH that use homogenates are called Northern blot and dot blot.
DNA microarrays Microarrays, also called DNA chips or gene chips, provide the newest and most dramatic improvement in gene technology. Because the nervous system exhibitstions of unlabeled antigen, which compete with the radioactively labeled antigen. The higher the concentration of unlabeled competitor antigen added, the lower the amount of radioactive antigen bound after the mixture has been incubated. The values are plotted as a standard curve and analyzed using appropriate computer software.
To determine how much of the antigen is in any experimental sample, other test tubes are prepared in just the same way except that the samples containing unknown amounts of antigen are added instead of the known antigen. By measuring the amount of radioactive antigen bound in the sample tubes compared to the standard curve, the amount of antigen in the sample can be calculated.
In situ hybridization In situ hybridization (ISH) makes it possible to locate cells in tissue slices that are manufacturing a particular protein or peptide in much the same manner that ICC identifies cells containing a particular protein. ISH is particularly useful in neuropharmacology for detecting the specific messenger RNA (mRNA) molecules responsible for directing the manufacture of the wide variety of proteins essential to neuron function, such as enzymes, structural proteins, receptors, ion channels, and peptides. Because the method detects cells with a precise RNA sequence, it is exceptionally specific and extremely sensitive. Besides locating cells containing specific mRNA, ISH is also used to study changes in regional mRNA levels after experimental manipulations. The amount of mRNA provides an estimate of the rate of synthesis of the particular protein. This means that if chronic drug treatment caused a decrease in enkephalin mRNA we could conclude that the protein the mRNA codes for has been down-regulated, that is, less of that protein is being synthesized.
As you recall from an earlier post, the double strands of DNA and corresponding mRNA have unique base-pair sequences responsible for directing the synthesis of a particular protein with its unique amino acid sequence. ISH depends on the ability to create probes by labeling single-stranded fragments of RNA made up of base-pair sequences complementary to those of the mRNA of interest. After the single strands are prepared, they are labeled radioactively or with dyes. When the tissue slices or cells are exposed to the labeled probe, the probe attaches (binds, or hybridizes) to the complementary base-pair sequences. After incubation, the tissue is washed and dehydrated before being placed in contact with X-ray film or being processed in other ways for visualization of cells containing the specific mRNA. The technique is extremely sensitive and can detect a very small number of cells that express a particular gene. If the researcher is interested only in measuring the amount of mRNA rather than visualizing its location, hybridization can be done using a tissue homogenate rather than a tissue slice. Two available methods of ISH that use homogenates are called Northern blot and dot blot.
DNA microarrays Microarrays, also called DNA chips or gene chips, provide the newest and most dramatic improvement in gene technology. Because the nervous system exhibits the greatest complexity of gene expression of all tissues, being able to examine all of the genes simultaneously can tell researchers which genes switch on and off together in response to a disease state, drug treatment, or environmental condition. One would assume that genes that increase or decrease their expression under the same condition probably work together to induce a cellular response. In addition, measuring the amount of various RNAs in a sample tells us both the types and amounts of proteins present. A study by Mirnics and colleagues (2000) demonstrated the technical elegance of microarray by identifying multiple presynaptic proteins that are underexpressed in the frontal lobes of schizophrenic individuals. Their results provide a predictive and testable model of the disorder.
The method is similar to that described for ISH, but rather than measuring a single mRNA, microarrays consist of between 1000 and 20,000 distinct complementary DNA sequences on a single chip (a structural support) of approximately thumbnail size. Each spot is only about 50 to 150 |J.m in diameter. This makes it possible to screen the expression of the entire genome of an organism in a single experiment on just a few chips. The tissue to be evaluated (for example, the frontal lobe from a schizophrenic individual compared to a normal frontal lobe) is dissected, and the mRNAs are isolated and labeled, then hybridized to the large number of immobilized DNA molecules on the chip. A scanner automatically evaluates the amount of hybridization of each of the thousands of spots on the chip, and computer analysis is used to identify the patterns of gene activity. Several excellent reviews of the microarray procedure and its application in areas such as aging, neuropharmacology, and psychiatric disorders are available (Luo and Geschwind, 2001; Marcotte et al. 2001).
New tools are used for imaging the struc-ture and function of the brain
Most conventional neurobiological techniques are designed to quantify or to localize significant substances in the nervous system. One of the greatest challenges in psychopharmacology has been to evaluate the functioning of the brain under varying conditions, particularly in the living human being. Advances in technology not only make the visualization of the CNS far more precise, but also provide the opportunity to visualize the functioning brain.
Autoradiography of dynamic cell processes You are already familiar with the technique of autoradiography for mapping cell components such as neurotransmitter receptors that have been radioactively labeled. Another important application of autoradiography is the tracing of active processes in the brain such as cerebral blood flow, oxygen consumption, local glucose utilization, or local rates of cerebral protein synthesis. 2-Deoxyglucose autoradiography is based on the assumption that when nerve cell firing increases, the metabolic rate, that is, the utilization of glucose and oxygen, also increases. By identifying cells that take up more glucose under experimental conditions such as drug treatment, we can tell which brain regions are most active. 2-Deoxyglucose (2-DG) is a modified form of the glucose molecule that is taken up by active nerve cells but is not processed in the same manner as glucose and remains trapped in the cell. If the 2-DG has been labeled in some way, the most active cells can be identified. The method involves injecting an animal with radioactive 2-DG before evaluating its behavior in a test situation. The experimenter then kills the animal, removes the brain, and slices it in preparation for autoradiography (described earlier). A similar (but nonlethal) technique can be performed with human subjects, using PET as described below.
A second way of identifying which brain cells are active is to locate cells that show increases in nuclear proteins involved in protein synthesis. The assumption is that when cells are activated, selected proteins called transcription factors (such as c-fos) dramatically increase in concentration over 30 to 60 minutes. The c-fos protein subsequently activates the expression of other genes that regulate protein synthesis. c-Fos can be located in the brain using ICC to stain cells with increased levels of the fos protein and hence increased cell activity.
Imaging techniques Since our ultimate goal is to understand how drugs affect the human brain and behavior, the most exciting advance in recent years has been the ability to visualize the living human brain. Although we can learn a lot by studying individuals with brain damage, until recently we could only guess at where the damage was located because the brain was not accessible until the individual died, often many years later. It was virtually impossible to know which specific brain area was responsible for the lost function. The human brain remained a bit of a “black box,” and our understanding of the neural processes responsible for human thinking and behavior were advanced primarily due to animal experiments. Because of recent advances in X-ray and computer technology, neuroscience can now not only safely visualize the detailed anatomy of the human brain but also identify the neural processes responsible for a particular mental activity. CT and MRI are techniques that create pictures of the human nervous system in far greater detail than previously possible with standard X-ray. Other techniques are designed to see functional activity in the human brain. These include PET, functional MRI, and computer-assisted electrical recording.
When standard X-rays are passed through the body, they are differentially absorbed depending on the density of the various tissues. Rays that are not absorbed strike a photographic plate, forming light and dark images. Unfortunately, the brain is made up of many overlapping parts that do not differ dramatically in their ability to absorb X-rays, so it is very difficult to distinguish the individual shapes of brain structures. Computerized tomography (CT) not only increases the resolution (sharpness of detail) of the image but also provides an image in three dimensions.
The individual undergoing a CT scan (sometimes called CAT scan, for computerized axial tomography) lies with his head placed in a cylindrical X-ray tube. A series of narrow, parallel beams of radiation are aimed through the tissue and toward the X-ray detectors. The X-ray source is rotated around the head while the detectors move on the opposite side in parallel. At each point of rotation, the source and detectors also move linearly. In this manner they make a series of radiation transmission readings, which is calculated by a computer and visually displayed as a “slice” through the brain. The slices can be reconstructed by the computer into three-dimensional images for a better understanding of brain structure.
Magnetic resonance imaging (MRI) further refines the ability to view the living brain by using computerized measurements of the distinct waves that different atoms emit when placed in a strong magnetic field and activated by radio-frequency waves. This method distinguishes different body tissues based on their individual chemical composition. Because tissues contain different amounts of water, they can be distinguished by scanning the magnetic-induced resonance of hydrogen. The image provides exquisite detail and, as is true for CT, sequential slices can be reconstructed to provide three-dimensional images.
It did not take long for scientists to realize the power of their new tool, and they proceeded to use the computerized scanning technique to view the localization of radioactively labeled materials injected into a living human. Positron emission tomography (PET) does not create images of the brain but maps the distribution of a radioactively labeled substance that has been injected into an individual. To do this safely with human subjects, we must use radioisotopes long half-lives, on the order of 1200 years for 3H or 5700 years for 14C, those used for PET have half-lives of 2 minutes (150), 20 minutes (nC), or 110 minutes (18F). Isotopes that decay and lose their radioactivity quickly (i.e., have a short half-life) emit positrons, which are like electrons but have a positive charge. When a positron expelled from the nucleus collides with an electron, both particles are annihilated and emit two gamma rays traveling in opposite directions. In a PET scanning device, detectors surround the head to track these gamma rays and locate their origin. The information is analyzed by computer and visualized as an image on the monitor.
PET is useful to neuropharmacology in several ways (Farde, 1996). First, a radioactively labeled drug or ligand can be administered and the location of binding in brain tissue can be seen. The technique has been used successfully to localize neurotransmitter receptors and identify where drugs bind. Perhaps even more exciting is the use of PET to determine which parts of the brain are active during the performance of particular tasks or cognitive problem solving. PET allows us to visualize brain activity, which is reflected in increases in glucose utilization, oxygen use, and blood flow, depending on which reagent has been labeled. Very much like autoradiography in living humans, PET can be used along with 2-DG to map brain areas that utilize increased glucose or demonstrate increased blood flow, both indicative of heightened neural activity.
Single-photon emission computerized tomography (SPECT) is very similar to PET imaging, but it is much simpler and less expensive since the radiolabeled probes do not have to be synthesized but are commercially available. When scanned, the radioactive compounds, either inhaled or injected, show the changes in regional blood flow. Although resolution is less accurate than with PET, the SPECT data can be combined with CT or MRI scans to localize the active areas more precisely than with SPECT alone.
Functional MRI (fMRI) has become the newest and perhaps most powerful tool in the neuroscientist’s arsenal for visualizing brain activity. To meet the increased metabolic demand of active neurons, the flow of blood carrying oxygen to these cells increases. Functional MRI can detect the increases in blood oxygenation caused by cell firing because oxygenated hemoglobin (the molecule that carries the oxygen in the blood and provides the red color) has a different magnetic resonance signal than oxygen-depleted hemoglobin. Functional MRI has several advantages over PET. First, MRI provides both anatomical and functional information in each subject and the detail of the image is far superior. Second, since the individual does not have to be injected with radioactive material, the measures can be made repeatedly to show changes over time. For the same reason, the procedure is essentially risk free, except for the occasional case of claustrophobia caused by the scanner. Third, the process is so rapid that brain activity can be monitored in real time (i.e., as it is occurring). In combination with recording electrical activity with electroencephalography (see the following paragraph), fMRI can produce three-dimensional images showing neural activity in interconnecting networks of brain centers. Temporal sequencing of information processing becomes possible, so one can see the changing locations of brain activity during tasks and cognitive processes. For an excellent introduction to brain imaging and its relationship to cognitive processes, refer to Posner and Raichle (1994).
Electroencephalography (EEG) In addition to improved visualization techniques and methods of mapping metabolic function in the human brain, a third non-invasive method of investigating human brain activity is now used often in neuropharmacology: electrical recording with electroencephalography (EEG). Electrodes are taped to the scalp in several locations, and the electrical activity that is recorded reflects the sum of electrical events of populations of neurons. Multiple electrades are used because a comparison of the signals from various locations can identify the origin of some waves. Although the method cannot identify specific cells that are active, it has been useful in studies of consciousness, sleep, and dreaming, as well as studies of seizure activity. Computer analysis of EEG signals can produce a color-coded map of brain electrical activity, which allows a visualization of electrical response to changing stimuli. Brain electrical activity mapping (BEAM) is one of the available display systems. Because EEG can detect electrical events in real time, it is very useful in recording electrical changes in response to momentary sensory stimulation; these changes are called event-related potentials or sensory evoked potentials. Evaluation of electrical responses in various clinical populations has led to improved understanding of attention deficits and processing differences in individuals with schizophrenia, Huntington’s disease, attention deficit disorder, and so forth.
Genetic engineering helps neuroscientists to ask and answer new questions
The excitement surrounding the completion of the Human Genome Project, in which all of the human genetic material has been mapped, has permeated both the scientific and the popular press. Although the term genetic engineering evokes both excitement and some trepidation in most people, the technology has at the very least provided amazing opportunities for neuroscience. Genetic engineering involves chemically modifying precise sites in the molecular structure of a gene in order to change the structure of the product produced by normal gene expression.
Targeted mutations, or knockout techniques This new method, based on advances in molecular biology, may represent the most sophisticated of all lesioning techniques yet described. With the ability to identify which piece of the chromosomal DNA (i.e., the gene) is responsible for directing the synthesis of a particular protein, neuroscience has the opportunity to alter that gene, causing a change in the expression of the protein. In essence, we are producing an animal model that lacks a particular protein (e.g., an enzyme, ion channel, or receptor) so that we can evaluate the post- lesioning behavior. We can also use these animals to identify the importance of that protein to specific drug effects.
The procedure requires elimination of the gene in isolated embryonic cells by destroying the base sequence on the chromosome that codes for a particular protein. The altered genes are then inserted into fertilized eggs of a foster mother. After birth, the pups are examined for incorporation of the altered DNA into their genes and for the possible expression of the mutation (e.g., altered behavior). As adults they are bred to create homozygous mice that lack the gene completely (knockout mice). Comparing the behavior and drug response of the altered mice with those of unaltered animals will tell us about the function of the protein that has been deleted. For neuropharmacologists, the protein of interest is often a receptor subtype or an enzyme that controls an important synthesizing or metabolizing process.
Gene replacement A second strategy involves the replacement of one gene for another, producing transgenic mice. As we learn more about the pathological genes responsible for neuropsychiatric diseases such as Huntington’s and Alzheimer’s diseases, it is possible to remove the human genes and insert them into mice to produce true animal models of the disorders. For an example, see the work by Carter and coworkers (1999), which measures motor deficits in mice transgenic for Huntington’s disease. With authentic animal models, neuroscience will be able to identify the cellular processes responsible for a disorder and develop appropriate treatments.
As is true for any revolutionary new technique, caution in interpreting the results is warranted. First, because behaviors are not regulated by single genes but by multiple interacting genes, changing or eliminating only one alters only a small part of the overall behavioral trait. Second, compensation by other genes for the missing or overexpressed gene may mask the functional effect of the mutation. Third, since the altered gene function occurs in all tissues at all stages of development, it is possible that changes in other organs or other brain areas are responsible for the behavioral changes. Finally, since these animals are developing organisms, environmental factors also have a significant effect on the ultimate gene expression. Several articles provide greater detail on the potential pitfalls of gene-targeting studies (Crawley, 1996; Gerlai, 1996; Lathe, 1996).
In addition to creating “mutant” animals, the genetic material can be inserted into cells (maintained in cell culture) that do not normally have a particular protein (e.g., receptor). The normal cell division process produces large numbers of identically altered cells, which we call cloning. These cells can then be used to screen new drugs using conventional pharmacological techniques for identifying agonists and antagonists.
A variation of gene modification uses short-term manipulations of the genetic material by intraventricular injection of antisense nucleotides that bind to targeted mRNAs, delay their translation, and increase their degradation. Such treatment produces a reversible “mutant” animal whose behavior or drug responsiveness can be evaluated. For instance, earlier research suggested that a decrease in the function of the neuropeptide called vasoactive intestinal peptide (VIP) in the hypothalamus (specifically the suprachiasmatic nucleus) may be responsible for the disturbances in circadian rhythms that occur during aging. To test this hypothesis, Harney et al. (1996) used antisense oligonucleotides that targeted VIP-con- taining neurons in the suprachiasmatic nucleus. The reduction in VIP concentration in the suprachiasmatic nucleus at different times after antisense administration. What the investigators found was that suppressing the synthesis of VIP in this brain region does indeed mimic the effect of age on cyclic hormone secretion. This technique is well suited to study the biological rhythm of reproductive hormones and their effects on behavior.
The goals of neuropsychopharmacology are to understand (1) the physiological and neurochemical mechanisms that are responsible for behavior as well as (2) how drugs interact with brain chemistry to modify that behavior. The tools and techniques of neuroscience allow us to combine results from studies using both humans and other animals. Lesioning selected brain areas using a stereotaxic device is the oldest of the methods, but modifications to this method that use neurotoxins to destroy cell bodies without damaging axons passing through the area have distinct advantages. Neurotoxins that are selective for a particular neurotransmitter provide the chance to lesion cells based on neurochemical identity. By implanting cannulas to deliver minute amounts of drugs, either agonists or antagonists, to functioning animals, we can test our knowledge of the role of specific receptors in behavior. Electrical stimulation and recording of the brain likewise provides a method to evaluate the role of particular cells in a behavioral response.
Emphasizing the role of receptors in pharmacology, the radioligand binding method has been developed to evaluate the number and affinity of specific receptor molecules. To locate these receptors more precisely in the brain, receptor autoradiography, both in vitro and in vivo, is used. The ability to make antibodies to various proteins paves the way for more precise cellular localization of receptors or other protein components of cells like enzymes. Immunocytochemistry uses the antibodies to precisely locate cells containing a particular protein, while a complementary technique, in situ hybridization, can tell us which cells are manufacturing a given molecule by labeling cells with an appropriate mRNA probe. DNA microarrays provide a means to simultaneously evaluate the expression of thousands of genes to identify those involved in complex clinical diseases along with potential therapeutics to combat the disorders.
It is now possible to visualize cognitive functioning in the human brain and use animals to examine the cellular details of that functioning. Computerized tomography and MRI provide detailed representations of the human brain. PET, SPECT, and MRI each provide a slightly different window into the working activity of the human brain using advanced computer technology to evaluate changes in cell function. Based on the premise that active brain cells use more glucose and oxygen and receive increased cerebral blood flow, the computerized methods are analogous to autoradiography but can be accomplished in an awake and functioning subject.
Clearly the use of genetic engineering to create transgenic or knockout mice provides the most sophisticated type of lesioning yet devised. By modifying a single piece of genetic material, the expression of a specific protein can be modified or eliminated to identify the biochemical and/or behavioral function of that protein.
Bear in mind that under normal circumstances several of these techniques are used in tandem to approach a problem in neuroscience from several directions. The power of these experimental tools is that when they are used together a more reasonable picture emerges and conflicting results can be incorporated into the larger picture. Only in this way can we uncover the neurobiological substrates of cognitive function and dysfunction. In every case, interpretation of these sophisticated approaches is subject to the same scrutiny that the earliest lesion experiments required. Remember, healthy skepticism is central to the scientific method.