During a lecture at a meeting of the Anthropological Society of Paris in 1861, Ernest Auburtin, a French physician, argued that language functions are located in the brain’s frontal lobes. Five days later a fellow French physician, Paul Broca, observed a brain-injured patient who had lost his speech and was able to say only “tan” and utter a swear word. The patient soon died. Broca and Auburtin examined the man’s brain and found the focus of his injury in the left frontal lobe.

By 1863 Broca had collected eight similar cases and concluded that speech is located in the third frontal convolution of the left frontal lobe—a region now called Broca’s area. Broca’s findings attracted others to study brain–behavior relationships in patients. The field that developed is what we now call neuropsychology, the study of the relations between brain function and behavior with a particular emphasis on humans. Today, measuring brain and behavior increasingly includes noninvasive imaging, complex neuroanatomical measurement, and sophisticated behavioral analyses.

At the beginning of the twentieth century, neuroanatomy’s primary tools were histological: brains were sectioned postmortem and the tissue (histo- in Greek) stained with various dyes. As shown in Figure 7-1, staining sections of brain tissue can identify cell bodies in the brain viewed with a light microscope (A), and selectively staining individual neurons reveals their complete structure (B). An electron microscope (C) makes it possible to view synapses in detail.

By 1905, light microscopic techniques allowed researchers such as Korbinian Brodmann to divide the cerebral cortex into many distinct zones based on the characteristics of neurons in those zones. Investigators presumed that cortical zones had specific functions. By the dawn of the twenty-first century, dozens of techniques had developed for labeling neurons and their connections, as well as glial cells. Contemporary techniques allow researchers to identify molecular, neurochemical, and morphological (structural) differences among neuronal types and ultimately to relate these characteristics to behavior. One fast-developing technique, shown in Figure 7-1D, uses a multiphoton microscope that makes it possible to image living brain tissue in three dimensions.

Connections between anatomy and behavior can be seen in studies of animals trained on various types of learning tasks, such as spatial mazes. Such learning can be correlated with a variety of neuronanatomical changes, such as modifications in the synaptic organization of cells in specific cortical regions—the visual cortex in animals trained in visually guided mazes is one example—or in the number of newly generated cells that survive in the hippocampus. The hippocampus is necessary, in mammals, for remembering the context in which we encounter information.

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Although changes in synaptic organization or cell survival have not yet been proved to be the basis of the new learning, experimental evidence reveals that preventing the growth of new hippocampal neurons leads to memory deficits. To test the idea that hippocampal neurons contribute to memory formation, researchers tested healthy rats and ADX rats—rats with adrenal glands removed, which eliminates the hormone corticosterone. Without corticosterone, neurons in the hippocampus die.

Procedure 1 in Experiment 7-1 contrasts the appearance of a healthy rat hippocampus (left) and neuronal degeneration in an ADX rat after surgery (right). The behavior of healthy and ADX rats was studied in the object–context mismatch task diagrammed in Procedure 2. During the training phase, the rats were placed in two distinct contexts, A and B, for 10 minutes on each of 2 days. Each context contained a different type of object. On the test day, the rats were placed in either context A or context B but with two different objects, one from that context and a second from the other context.

As noted in the Results section of the experiment, when healthy rats encounter objects in the correct context, they spend little time investigating because the objects are familiar. If, however, they encounter an object in the wrong context, they are curious and spend about three-quarters of their time investigating, essentially treating the mismatched object as new. But the ADX rats with hippocampal cell loss treated the mismatched and in-context objects the same, spending about half of their investigation time with each object.

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Another group of ADX rats given treatment known to increase neuron generation in the hippocampus—enriched housing and exercise in running wheels—was unimpaired at the context mismatch task. Experiment 7-1 concludes that cellular changes in the hippocampus and behavioral changes are closely linked: hippocampal neurons are necessary for contextual learning.

The brain’s ultimate function is to produce behavior (movement). It follows that brain dysfunction should alter behavior in some way. The study of brain–behavior relationships in both humans and laboratory animals is behavioral neuroscience, the study of the biological bases of behavior.

A major challenge to behavioral neuroscientists is developing methods for studying both typical and atypical behavior. Measuring behavior in humans and laboratory animals differs in large part because humans speak: investigators can ask them about their symptoms. It is also possible to use both paper-and-pencil and computer-based tests to identify specific symptoms in people.

Measuring behavior in laboratory animals is more complex. Researchers must learn to speak “ratese” with rat subjects or “monkeyese” with monkeys. In short, researchers must develop ways to enable the animals to reveal their symptoms. The development of the fields of animal learning and ethology, the objective study of animal behavior, especially under natural conditions, provided the basis for modern behavioral neuroscience (see Whishaw & Kolb, 2005). To illustrate the logic of behavioral neuroscience, we describe some measurement tools used with humans and some used with rats.

Neuropsychological Testing of Humans

The brain has exquisite control of an amazing array of functions ranging from movement control and sensory perception to memory, emotion, and language. As a consequence, any analysis of behavior must be tailored to the particular function(s) under investigation. Consider the analysis of memory.

People with damage to the temporal lobes often complain of memory disturbance. But memory is not a single function; rather, multiple as well as independent memory systems exist. We have memory for events, colors, names, places, and motor skills, among other categories, and each must be measured separately. It would be rare indeed for someone to be impaired in all forms of memory. Neuropsychological tests of three distinct forms of memory are illustrated in Figure 7-2.

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The Corsi block-tapping test shown in Figure 7-2A requires participants to observe an experimenter tap a sequence of blocks—blocks 4, 6, 1, 8, 3, for instance. The task is to repeat the sequence correctly. The subject does not see numbers on the blocks but rather must remember the location of the tapped blocks.

The Corsi test provides a measure of short-term recall of spatial position, an ability we can call block span. The test can be made more difficult by determining the maximum block span of an individual (say, 6 blocks) and then adding one (span + 1). By definition, the participant will fail on the first presentation, but given the span + 1 repeatedly, will eventually learn it.

Span + 1 identifies a different form of memory from block span. Different types of neurological dysfunction interfere differentially with tasks that superficially appear quite similar. Block span measures the short-term recall of information, whereas the span + 1 task reflects the learning and longer-term memory storage of information.

The mirror-drawing task (Figure 7-2B) requires a person to trace a pathway, such as a star, by looking in a mirror. This motor task initially proves difficult because our movements appear backward in the mirror. With practice, participants learn how to accomplish the task accurately, and they show considerable recall of the skill when retested days later. Curiously, patients and subjects with certain types of memory problems have no recollection of learning the task on the previous day but nevertheless perform it flawlessly.

In the recency memory task (Figure 7-2C), participants are shown a long series of cards, each bearing two stimulus items that are words or pictures. On some trials a question mark appears between the items. The subjects’ task is to indicate whether they have seen the items before and if so, which item they saw most recently. People may be able to recall that they have seen items before but be unable to recall which was most recent. Conversely, they may not be able to identify the items as being familiar, but when forced to choose the most recent one, they can identify it correctly.

The latter, counterintuitive result reflects the need for behavioral researchers to develop ingenious ways of identifying memory abilities. It is not enough simply to ask people to recall information verbally, although this too measures a form of memory.

Behavioral Analysis of Rats

Over the past century, psychologists interested in the neural basis of memory have devised a vast array of mazes and other tests and tasks to investigate different forms of memory in laboratory animals. Figure 7-3 illustrates three tests based on a task devised by Richard Morris in 1980. Researchers place rats in a large swimming pool where an escape platform lies just below the water surface, invisible to the rats.

In one version of the task, place learning, the rat must find the platform from any starting location in the pool (Figure 7-3A). The only cues available are outside the pool, so the rat must learn the relation between several cues in the room and the platform’s location. In a second version of the task, matching-to-place learning, the rat has already learned that a platform always lies somewhere in the pool but is moved to a different location every day. The rat is released and searches for the platform (Figure 7-3B). Once the rat finds the platform, the animal is removed from the pool and after a brief delay (such as 10 seconds) is released again. The rat’s task is to swim directly to the platform.

The challenge for the rat in the matching-to-place test is to develop a strategy for finding the platform consistently: it is always in the same location on each trial each day, but each new day brings a new location. In the landmark version of the task, the platform’s location is identified by a cue on the pool wall (Figure 7-3C). The platform moves on every trial, but the relation to the cue is constant. In this task the brain is learning that the distant cues outside the pool are irrelevant; only the local cue is relevant. Rats with different neurological perturbations are selectively impaired in the three versions of the swimming pool task.

Another type of behavioral analysis in rats is related to movement. A major problem facing people with stroke is a deficit in controlling hand and limb movements. The prevalence of stroke has prompted considerable interest in devising ways to analyze such motor behaviors for the purpose of testing new therapies for facilitating recovery. Ian Whishaw (Whishaw & Kolb, 2005) has devised both novel tasks and novel scoring methods to measure the fine details of skilled reaching movements in rats.

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In one test, rats are trained to reach through a slot to obtain a piece of sweet food. The movements, which are remarkably similar to the movements people make in a similar task, can be broken down into segments. Investigators can score the segments separately, as they are differentially affected by different types of neurological perturbation.

The photo series in Figure 7-4 details how a rat orients its body to the slot (A), puts its hand through the slot (B), rotates the hand horizontally to grasp the food (C), then rotates the hand vertically and withdraws it to obtain the food (D). Contrary to reports common in neurology textbooks, primates are not the only animals to make fine digit movements, but because the rat’s hand is small and moves so quickly, digit dexterity can be seen in rodents only with use of high-speed videography.

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One strategy for studying brain–behavior relationships is to manipulate some aspect of brain function and see how behavior changes. Investigators do so to develop hypotheses about how the brain affects behavior, then to test those hypotheses.

Neuroscientists hypothesize about the functions of brain regions by studying how removing or inhibiting them affects behavior. Broca studied patients with naturally occurring injuries and made inferences about language organization. Similarly, Parkinson patients have motor disturbances and associated cell death in the substantia nigra (Figure 7-5). It is a small step experimentally to injure specific brain regions in laboratory animals, then study their behavior. Such studies tell investigators not only about the injured region’s function but also what the remaining brain can do in the absence of the injured region. Neuroscientists also study brain region functions to form hypotheses, for example, on how exciting a region affects behavior. Certain drugs and stimulation techniques that increase brain region activity result in behavioral change.

A second reason to manipulate the brain is to develop animal models of neurological and psychiatric disorders. The general presumption in neurology and psychiatry is that it ought to be possible to restore at least some healthy functioning by pharmacological, behavioral, or other interventions. A major hurdle for developing such treatments is that, like most new medical treatments, they must be tested in nonhuman subjects first. (In Section 7-7, we take up scientific and ethical issues surrounding the use of animals in research.)

For brain disorders, researchers must develop models of diseases before they can be treated. Consider dementia, a progressive memory impairment that appears to be related to neuronal death in specific brain regions. The goal for treatment is to reverse or prevent cell death, but developing effective treatments requires an animal model that mimics dementia.

Brains can be manipulated in various ways, the precise manner depending on the specific research question being asked. Researchers can manipulate the whole animal by exposing it to differing diets, social interactions, exercise, sensory stimulation, and a host of other experiences. For brain manipulation, the principal direct techniques are to inactivate the brain via lesion or with drugs or to activate it with electrical stimulation, drugs, or light.

Brain Lesions

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The first—and the simplest—technique used was to ablate (remove or destroy) tissue. Beginning in the 1920s, Karl Lashley, a pioneer of neuroscience research, used ablation, and for the next 30 years he tried to find the site of memory in the brain. He trained monkeys and rats on various mazes and motor tasks, then removed bits of cerebral cortex with the goal of producing amnesia for specific memories.

To his chagrin, Lashley failed in his quest. He observed instead that memory loss was related to the amount of tissue he removed. The only conclusion Lashley could reach was that memory is distributed throughout the brain and not located in any single place. Subsequent research strongly indicates that specific brain functions and associated memories are indeed localized to specific brain regions. Ironically, just as Lashley was retiring, William Scoville and Brenda Milner (1957) described a patient from whose brain Scoville had removed both hippocampi as a treatment for epilepsy. The surgery rendered this patient amnesic. During his ablation research, Lashley had never removed the hippocampi because he had no reason to believe the structures had any role in memory. And because the hippocampus is not accessible on the brain’s surface, other techniques had to be developed before subcortical lesions could be used.

The solution to accessing subcortical regions is to use a stereotaxic apparatus, a device that permits a researcher or a neurosurgeon to target a specific part of the brain for ablation, as shown in Figure 7-6. The head is held in a fixed position, and because the location of brain structures is fixed in relation to the junction of the skull bones, it is possible to visualize a three-dimensional brain map.

Rostral–caudal (front to back) measurements, corresponding to the x-axis in Figure 7-6, are made relative to the junction of the frontal and parietal bones (the bregma). Dorsal–ventral (top to bottom) measurements, the y-axis, are made relative to the surface of the brain. Medial–lateral measurements, the z-axis, are made relative to the midline junction of the cranial bones. Atlases of the brains of humans and laboratory animals have been constructed from postmortem tissue so that the precise location of any structure can be specified in three-dimensional space.

Consider the substantia nigra. To ablate this region to induce a rat to display symptoms of Parkinson disease, the structure and its three-dimensional location in the brain atlas is located. A small hole is then drilled in the skull, as shown in Figure 7-6, and an electrode lowered to the substantia nigra. If a current is passed through the electrode, the tissue in the region of the electrode tip is killed, producing an electrolytic lesion.

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A problem with electrolytic lesions: not only are the neurons of the targeted tissue killed but so are any nerve fibers passing through the region (in this case, substantia nigra). One solution is to lower a narrow metal tube (a cannula) instead of an electrode, infuse a neuron-killing chemical, and thus produce a neurotoxic lesion. (Figure 7-22 diagrams this procedure.) A selective toxin can be injected that kills only neurons, sometimes only certain types of neurons, and spares the fibers.

To make a rat parkinsonian, a toxin can be injected that is selectively taken up by dopaminergic neurons; this leads to a condition that mimics human Parkinson pathology. Animals with such neurotoxic lesions have a variety of motor symptoms including hypokinesia (slowness or absence of movement), short footsteps, and tremor. Drugs such as L-dopa, an agonist that enhances dopamine production, and atropine, an antagonist that blocks acetylcholine production, relieve these symptoms in Parkinson patients. Ian Whishaw and his colleagues (Schallert et al., 1978) thus were able to selectively lesion the substantia nigra in rats to produce a behavioral model of Parkinson disease.

The invasive techniques described so far result in permanent brain damage. With time, the research subject will show compensation, the neuroplastic ability to modify behavior from that used prior to the damage. To avoid compensation following permanent lesions, researchers have also developed temporary and reversible lesion techniques such as regional cooling, which prevents synaptic transmission. A hollow metal coil is placed next to a neural structure; then chilled fluid is passed through the coil, cooling the brain structure to about 18ºC (Lomber & Payne, 1996). When the chilled fluid is removed from the coil, the brain structure quickly warms, and synaptic transmission is restored. Another technique involves local administration of a GABA agonist, which increases local inhibition and in turn prevents the brain structure from communicating with other structures. Degradation of the GABA agonist reverses the local inhibition and restores function.

Brain Stimulation

The brain operates on both electrical and chemical energy, so it is possible to selectively turn brain regions on or off by using electrical or chemical stimulation. Wilder Penfield, in the mid-twentieth century, was the first to use electrical stimulation directly on the human cerebral cortex during neurosurgery. Later researchers used stereotaxic instruments to place an electrode or a cannula in specific brain locations. The objective: enhancing or blocking neuronal activity and observing the behavioral effects.

Perhaps the most dramatic research example comes from stimulating specific regions of the hypothalamus. Rats with electrodes placed in the lateral hypothalamus will eat whenever the stimulation is turned on. If the animals have the opportunity to press a bar that briefly turns on the current, they quickly learn to press the bar to obtain the current, a behavior known as electrical self-stimulation. It appears that the stimulation is affecting a neural circuit that involves both eating and pleasure.

Brain stimulation can also be used as a therapy. When the intact cortex adjacent to cortex injured by a stroke is stimulated electrically, for example, it leads to improvement in motor behaviors such as those illustrated in Figure 7-4. Cam Teskey and his colleagues (Brown et al., 2011) successfully restored motor deficits in a rat model of Parkinson disease by electrically stimulating a specific brain nucleus.

Deep-brain stimulation (DBS) is a neurosurgical technique. Electrodes implanted in the brain stimulate a targeted area with a low-voltage electrical current to facilitate behavior. DBS to subcortical structures—for example, the globus pallidus in the basal ganglia of Parkinson patients—makes movements smoother. Medications can often be reduced dramatically. DBS using several neural targets is an approved treatment for obsessive-compulsive disorder. Experimental trials are underway to identify the brain regions optimal for DBS to be used as a treatment for intractable psychiatric disorders such as major depression (Schlaepfer et al., 2013), schizophrenia, and possibly for epilepsy; also for stimulating recovery from TBI.

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Electrical stimulation of the brain is invasive: holes must be drilled in the skull and an electrode lowered into the brain. Researchers have taken advantage of the relation between magnetism and electricity to develop a noninvasive technique, transcranial magnetic stimulation (TMS). A small wire coil is placed adjacent to the skull, as illustrated in Figure 7-7A. A high-voltage current pulsed through the coil produces a rapid increase and subsequent decrease in the magnetic field around the coil. The magnetic field easily passes through the skull and causes a population of neurons in the cerebral cortex to depolarize and fire (Figure 7-7B).

If the motor cortex is stimulated, movement is evoked, or if a movement is in progress, it is disrupted. Similarly, if the visual cortex is stimulated, the participant sees dots of light (phosphenes). The effects of brief pulses of TMS do not outlive the stimulation, but repetitive TMS (rTMS), or continuous stimulation for up to several minutes, produces more long-lasting effects. TMS and rTMS can be used to study brain–behavior relationships in healthy participants, and rTMS has been tested as a potential treatment for a variety of behavioral disorders. A growing body of research also supports its antidepressant actions.

Drug Manipulations

Brain activity can also be stimulated by administration of drugs that pass into the bloodstream and eventually enter the brain or, through an indwelling cannula (illustrated in Figure 7-22), that allows direct application of them to specific brain structures. Drugs can influence the activity of specific neurons in specific brain regions. For example, the drug haloperidol, used to treat schizophrenia, reduces dopaminergic neuron function and makes healthy rats dopey and inactive (hypokinetic).

In contrast, drugs that increase dopaminergic activity, such as amphetamine, produce hyperkinetic rats—rats that are hyperactive. The advantage of administering drugs is that their effects wear off in time as the drugs are metabolized. It thus is possible to study drug effects on learned behaviors, such as skilled reaching (see Figure 7-4), and then to reexamine the behavior after the drug effect wears off.

Claudia Gonzalez and her colleagues (2006) administered nicotine to rats as they learned a skilled reaching task, then studied their later acquisition of a new skilled reaching task. The researchers found that the earlier nicotine-enhanced motor learning impaired the later motor learning. This finding surprised the investigators, but it now appears that repeated exposure to psychomotor stimulants such as amphetamine, cocaine, and nicotine can produce long-term effects on the brain’s later plasticity, its ability to change in response to experience, including learning specific tasks.

Optogenetics and Chemogenetics

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Within the span of a decade, synthetic biology, the design and construction of biological devices, systems, and machines not found in nature, has transformed how neuroscientists manipulate brain cells. Techniques include inserting a genetic sequence into the genome of a living organism. The transgenic technique of optogenetics, for example, combines genetics and light to control targeted cells in living tissue. A sequence that codes for a light-sensitive protein associated with an ion channel enables investigators to use light to change the shape (conformation) of the channel.

Optogenetics is based on the discovery that light can activate certain proteins that occur naturally and have been inserted into cells of model organisms. For example, opsins, proteins derived from microorganisms, combine a light-sensitive domain with an ion channel. The first opsin used for the optogenetic technique was channelrhodopsin-2 (ChR2). When ChR2 is expressed in neurons and exposed to blue light, the ion channel opens and immediately depolarizes the neuron, causing excitation. In contrast, stimulation of halorhodopsin (NpHR) with a green-yellow light activates a chloride pump, hyperpolarizing the neuron and causing inhibition. A fiber-optic light can be delivered to selective brain regions such that all genetically modified neurons exposed to the light respond immediately (Haubensak et al., 2010).

Optogenetics has tremendous potential as a research tool. Investigators can insert light-sensitive proteins into specific neuron types, such as pyramidal cells, and use light to selectively activate a single cell type. Researchers hail optogenetics for its high spatial and temporal (time) resolution. Ion channels can be placed into specific cell lines and turned on and off on millisecond time scales. Optogenetics also finds application in behavioral studies. Within the limbic system, for example, the amygdala is a key structure in generating fear in animals. If it is targeted with opsins then exposed to an inhibitory light, rats immediately show no fear and wander about in a novel open space. As soon as the light is turned off, they scamper back to a safe hiding place.

In the transgenic technique, chemogenetics, the inserted synthetic genetic sequence codes for a G protein–coupled receptor engineered to respond exclusively to a synthetic small-molecule “designer drug.” Chemogenetics is best known by the acronym DREADD (designer receptor exclusively activated by designer drugs). Its principle advantage is that the drug will activate only the genetically modified receptors, and the receptors will be activated only by the designer drug, not by endogenous molecules (Wess et al., 2013). Thus, specificity is high, but temporal resolution is much lower than with optogenetics because receptors are activated by drugs rather than by light.

Measuring and Manipulating Brain and Behavior

Before you continue, check your understanding.

Question 1

Question 2

Question 3

Question 4

Answers appear in the Self Test section of the book.