Custom-Made Neural Stem Cells

It is ironic that an attempt to do a molecular end-run around a politically hot topic could result in an important breakthrough in the treatment of neurological disease with potentially strong implications for the psychiatric community. Ironic maybe, but true.

In this column, we explore how the judicious use of neural stem cells (NSCs) has led to a research Holy Grail: the creation of research-ready, patient-specific neurons. This technology did not use the famously controversial embryonic stem cells. These custom-made NSCs were created from politically neutral adult tissues (fibroblasts), which were originally isolated from an affected patient. With no embryo in sight, scientists genetically reprogrammed fibroblasts into stem cells, which were then induced to develop into NSCs. This is an extraordinary finding with many topics to be discussed here:

• The potential research utility for patient-specific neurons

• An explanation of how stem cells can be made from adult tissues

• A striking set of results that involve one of the most commonly inherited and lethal childhood neurological disorders: spinal muscular atrophy (SMA)

Research utility for NSCs

Of what possible utility could molecular investigations of a motor disorder have for the mental health community? Before getting into the specifics of the breakthrough, it might be useful to address a real-world psychiatric need, using depression and SSRIs as an example, to see where these data fit.

When we consider the molecular mechanisms of SSRI interactions, it is easy to resort to commonly taught ideas about interactions that involve a single synapse. Nothing could be further from the truth. The most comprehensive neurological view of SSRI interactions must take into account the participation of thousands of individual neurons strung together in coordinated, complex neural networks.

And not just serotonergic neurons. These cells are in contact with many other central nervous denizens, from adjacent glial cells to the extracellular matrix into which the cells are embedded. What do these circuits actually look like in patients who are vulnerable to depression? Is their architecture all that different from patients who do not exhibit this vulnerability? If there are differences, could they eventually predict drug efficacy? Could these differences only be detected by constructing parts of the circuit from scratch, or could they be observed at the level of a single cell?

The first step in answering these questions involves growing a custom-made batch of serotonergic neurons derived only from the affected patients, and then asking relevant structure/function questions. From attempting to understand molecular mechanisms of disease to testing the efficacy of potential medications, such patient-specific test beds would have a powerful research utility. Until recently, the creation of such tailor-made neural substrates had been an impossible goal.

While it will certainly be quite some time before we can grow entire parkinsonian dopaminergic pathways in a dish, it is now possible to create individual patient-specific neurons in culture. The technology comes from that end-run I mentioned earlier, through the use of a certain type of stem cell. It is to these interesting cellular substrates that we now turn.

Inducible stem cells

To say that embryonic stem cell research has been subject to heated political debate is an understatement. The bugaboo has been the source materials from which the stem cells would be isolated—human embryos—many left over from embryos generated in in vitro fertilization laboratories.

In 2006, researchers found a way to create stem cells that bypassed the need for human embryos. The original technique involved the introduction of 4 specific gene products into mature mouse fibroblasts. Surprisingly, this cocktail was found to reprogram adult stem cells and reverse-engineer them into pluripotent stem cells. Like embryonic stem cells, the altered stem cells had the ability to differentiate into any cell type. Eventually, a protocol was developed that did the same thing in human tissues. The cells were called iPSCs, short for induced pluripotent stem cells.

This was quite a breakthrough. No longer would researchers need to harvest cells from extant human embryos to do stem cell research. Skin cells would do. Scientists were soon able to regenerate—and then correct—molecular dysfunction in a mouse model of sickle cell anemia using this technology.

Could any of this work apply to humans, specifically to human neural tissue? Another successful round of experiments (with amyotrophic lateral sclerosis neurons) prompted researchers to study motor disease, ie, SMA.

Of those hereditary neurological disorders capable of causing death in pediatric populations, SMA is easily the most common. The disease is unique to humans and associated with 2 genes, SMN1 and SMN2. For reasons that are not well understood, the absence of the survival motor neuron (SMN) protein results in an alteration of the function of spinal motor neurons. The primary feature is muscle weakness and atrophy. Death occurs at infancy in the most severe forms of the disease, with symptoms generally presenting several weeks after birth. There are many other, nonlethal forms of the disorder, however, with a wide spectrum of symptoms that range from trivial motor effects to catastrophic impairment.

Why this variation? Both genes express in unaffected individuals, but the biological heavy lifting belongs to the SMN1 gene. Because of structural constraints, the expression pattern of the SMN2 gene normally results in only 10% of its protein being processed as a full-length (and functional) polypeptide; 90% of its protein output is truncated (and nonfunctional). That is okay, as long as the SMN1 gene is intact. But when SMN1 is mutated and silent, the disease condition results. Assuming there is a damaged SMN1, the severity of SMA varies according to the number of other SMN2 copies the infant may carry. The more copies of SMN2 gene, the greater the population of functional protein. This interaction explains in part why there can be so much varia-tion in the clinical presentation.

The great mystery is why SMN protein loss results in motor cell alterations that lead to the disease state in the first place. The protein is known to be essential for normal messenger RNA processing and is expressed throughout the body. Yet its absence most severely affects spinal motor neurons.

The most exacting way to attack this “black box” would be to isolate the motor neuron populations from the patient, then compare these populations with unaffected controls and look for differences, of which there are many. These include responses to various medications. It is well known that the application of valproic acid (an anticonvulsant and/or mood stabilizer) or tobramycin(Drug information on tobramycin) (an aminoglycoside) to cultured cells, for example, leads to changes in the expression patterns of both full-length SMN protein and truncated forms. What is the molecular basis of this unusual interaction? And could such differences be used as a “molecular flashlight” to ferret out other secrets regarding the SMN protein? Creating custom-made neurons—one population from an affected individual, another from an unaffected control—would certainly give a test bed capable of answering this question.

The data

Studying these 2 populations is precisely what a group of investigators did. The researchers isolated fibroblasts from an affected child and also from the child’s healthy unaffected mother.

The next step was to generate custom-made neurons. Several steps would be required (Figure). First, using the iPSC protocols I mentioned, the researchers would attempt to create stem cells from both child and parent sources. If that worked, the researchers would then try to induce these patient-derived stem cells into motor neurons—ones that would carry the same biological mechanisms observed in both the diseased and the healthy populations. If successful, the researchers would have their custom-made test beds. They could begin characterization studies; reactions to valproic acid and tobramycin would make obvious first choices to try.

The first step worked. The researchers were able to generate custom-made stem cells from both child and parent. The researchers then tackled the hard part: manufacturing spinal motor neurons from these stem cell populations. They certainly generated promising cellular populations. But the iPSC technolo-gies are new enough that a visual inspection of the generated cells might be necessary—but certainly not sufficient—to show the presence of motor neurons.

There are ways to gain greater reassurance. One way to assay the success of the protocol is to look for bona fide molecular markers of developing spinal neurons. It is known, for example, that extant motor neurons express the protein SMI032 and choline acetyltransferase. Did these induced cells express such proteins? The answer turned out to be yes, both for the affected child and for the unaffected parent. Developing cells in these populations possess transcription factors such as HOXB4, ISLET1, HB9, and OLIG2 as well. Did the induced populations express these markers? They did indeed. While not completely conclusive, it appeared that the researchers had generated patient-specific motor neurons from known affected and unaffected sources.

The next characterization experiments also yielded fruit. They were able to find that the child and parent neurons reacted very differently to the normally stimulating effects of valproic acid and tobramycin. The child’s cells showed elevated levels of SMN protein, both of the truncated form and full-length version. In addition, SMN-containing nuclear structures were altered. No such elevation occurred in the unaffected maternal line of cells.

These differences were significant for 2 reasons. First, it gave the investigators a toehold in their attempts to characterize at a more intimate level the differences between affected and unaffected cells. Second, the differences were discovered as reactions to known medications. The hope is that similar approaches could be used to test the efficacy of various medications before committing to human trials.

Conclusions

These data, full of promising implications as they are, need to be treated with some caution. First, the experimental cells are pure populations derived from stem cells. This hardly reflects the physical in vivo situation. The cells and matrix components that normally surround such cells in nature, including skeletal muscle tissues and even other neurons, are not present in these studies.

Another objection concerns the fidelity of the conversion process itself. The differentiation pattern seen in various molecular markers hinted that the investigators generated real live spinal motor neurons; however, one cannot a priori say they have in every way created a motor neuron that precisely mimics the real-world situation. These cells may lack many subtle molecular processes—and a few extra, equally subtle interactions—that could easily escape detection, at least by current technologies. Because subtle differences can profoundly influence intracellular molecular interactions, especially when we think about reactions to medications, this is a true concern.

The most exciting aspect of these studies comes from what the future holds. A great deal of speculation has gone into thinking about how to tailor medications to individual patients. That certainly is a psychiatric issue . . . I need not talk to this audience about the variable effects of, say, fluoxetine(Drug information on fluoxetine) on clinical outcomes. We have visited this topic in past columns. The ability to create patient-specific cellular test beds may go a long way toward solving some of these problems. Indeed, clinics of the future might routinely screen to decide what medications their patients should receive—and in what concentrations.

There is much work to do. To date, none has been applied to neurological systems relevant to mental health professionals. Even given the cautions mentioned above, there is no reason why it couldn’t. That’s not bad for having to do with an end-run around a hostile, politically charged issue such as stem cell debates. Would that all ethical issues could be decided so cleanly, or with so much fruit.

This article first appeared in Psychiatric Times.

To Sirtuin With Love: Caloric Restrictions and the Genes of the Aging Brain

One of the most interesting research efforts of the past few years seems to have taken a page from the preoccupations of the self-help magazine world: the cognitive decline of the brains of aging baby boomers and the obese nature of their grandchildren. These topics have been united by the well-established finding that restricting caloric intake leads to an increased life span in almost every animal tested. Because eating habits are formed early in life, the prediction is that a sensible diet will increase a person’s life span.

Is that true? The link between caloric restriction and life span is certainly solid, although the jury is still out on many of the claims made in the general media. Serious scientists have been studying the molecular biology behind this link for a number of years. One particular group of sequences seems to stand in the gap between aging and food, that of the sirtuin family of genes (also called silent information regulator [SIR] genes). One member of the family, the SIR2 gene, has been studied in particular detail. This article discusses age-related cognitive decline, caloric intake restriction, and the role SIR2 plays in the process.

As the brain ages

It is the canonical experience of people older than 40 that senior moments become an increasingly familiar part of one’s thinking life. We forget names, we forget places, we forget where we put our car keys, and we wonder when—and why—our retrieval systems began to abandon us. Higher-order processing begins to change as well as memory, perhaps not as obviously, but apparently in a far more dramatic way. Although the types of cognitive decline clearly vary from one person to the next, no one escapes these behavioral changes completely or, perhaps, the panic that ensues for some individuals when they compare their previous talent with current abilities.

It is axiomatic that cognitive decline occurs because of physical changes that human brains undergo as they age. Yet demonstrating the specifics of the relationship has not always been easy. With the advent of more sophisticated technologies (and improvements in older technologies), that has begun to change. We now know that changes in the regulation of global gene expression patterns—mostly down-shifting—are observed in a broad swath of the CNS during aging. Gene products specifically involved in the physiological processing of inhibitory signals mediated by γ-aminobutyric acid) have shown particular vulnerability. These neuron-related changes are disproportionately large when compared with changes in other tissues (eg, kidney and muscle tissues show age-related up-regulation).

Alterations in gross neuroanatomical structure have also been observed for many years. Some of the most dramatic involve disruptions of the myelinated fibers that yoke disparate brain regions together to provide specific functions—particularly in the prefrontal cortex (PFC). The changes are generally not due to neuronal loss in the PFC, which is actually quite minimal in most aging brains, but to loss of functional connectivity. The accompanying disruptions of neural integration in these regions of the aging brain result in less organized activity than is found in the brains of youthful controls. Such alterations are thought to be associated with a measurable behavioral change in aging populations: disruption of executive function (a task involved in everything from impulse control to planning for the future). This is part of a general age-related decline in the brain’s higher-order functional abilities.

One of the challenges of studying cognitive decline in elderly populations is separating normal changes in cognition from abnormal pathological changes. Although these are not always easy to distinguish, examination of the aging hippocampus has provided valuable insights. The normal aging pattern of the hippocampus involves an inhibition of metabolic activities of the dentate gyrus and subiculum. That is not what you see in patients who have Alzheimer disease. At least initially, the inhibition primarily targets the entorhinal cortex. Neuronal death in these tissues, with a general volumetric shrinkage of the medial temporal lobe, has been shown to distinguish the disease state from typical aging processes.

It is also a matter of calories

Many of the data presented above appear to describe natural, typical processes. But are they inevitable? One of the first questions many people ask after going a few rounds with their senior moment brains is: can the decline be reversed? These are often the questions asked by people who want to increase their life span. The surprising answer to both questions, in a few cases described below, is yes.

One of the most remarkable discoveries in the field of life span alteration occurred in the past century and has to do with caloric restriction. (This does not mean caloric starvation; malnutrition does not provide the benefit and is a completely different issue.) A controlled decrease in the amount of calories consumed has changed the life span of a surprising variety of animals, including mammals. It truly does mean that if you eat less, you will live longer.

The benefits of caloric restriction have been shown to have brain-specific effects as well. Caloric restriction can alter the regulatory genetic down-shifting phenomenon discussed earlier. It has also been shown to change the age-related neuronal degradation in nonhuman primates. Most relevant to our story, caloric restriction can affect human cognitive functioning. In one remarkable study, a caloric restriction protocol that lasted 90 days dramatically improved the verbal memories of a healthy geriatric cohort. Caloric restriction has even been shown to inhibit amyloid-related plaque formation in transgenic mice models of Alzheimer disease.

A matter of genes

Such robust findings work like scaffolding for researchers interested in the molecular biology behind the aging process and have certainly piqued the interest of people wanting to extend their life spans. What is the molecular mechanism behind the life span–extending properties of feeling hungry all the time? One of the earliest fruits of these research efforts was the isolation and characterization of the SIR genes.

The SIR genes were first isolated and characterized in yeast. They are a highly conserved family of sequences found in animals as diverse as roundworms and humans. One intensely studied member of this family is the SIR2 gene (Figure).

The SIR2 gene product functions as an NAD+-dependent deacetylase. In the presence of NAD+, SIR2 removes acetyl groups from proteins. Histone proteins are a favorite target of SIR2. As you may recall from your undergraduate days, histones are groups of proteins around which DNA molecules wrap themselves, somewhat like popcorn wrapped around string. Histones are deeply involved in regulating gene expression. Adding or subtracting subgroups to histones can profoundly influence the expression patterns of the genes in contact with the molecules.

How does SIR2 fit into the caloric restriction story? It was shown years ago that if you introduce SIR2 into yeast in such a fashion that you overdrive its expression, you can increase the yeast’s life span (measured as the number of times a cell completes a round of replication). If you severely restrict the caloric intake of an unmanipulated yeast, you can do the same thing. If you look for levels of SIR2 protein, you find that starvation has elevated its activity. The association appears to be strong, both by correlation and by direct intervention. The link between SIR2 protein and caloric restriction was first found in insects and then in mammals.

Subsequent research has muddied the waters of this otherwise seemingly tight story, however. Other researchers failed to replicate the results of the initial findings. Questions about various technical aspects that provided the original findings have been raised as well. There appear to be differences between genetic backgrounds in the primary test vehicles, from yeast to mice. These issues have yet to be fully resolved.

Such controversies are the nature of a good research project that, while hardly finished, has reached a certain maturity. These controversies are not deal killers regarding the association between the aging process and what goes in your mouth. The devil, as they say, is in the details. The fact that these issues can be raised at all demonstrates the enormous strides that researchers are making regarding the association between aging and eating—two of the most socially important issues of our time. That the arguments can revolve around deciding how the subtraction of acetyl groups changes the cell cycle simply shows how intimate, and how sophisticated, the progress has become.

This article originally appeared in Psychiatric Times.

References

Andrews-Hanna JR, Snyder AZ, Vincent JL, et al. Disruption of large-scale brain systems in advanced aging. Neuron. 2007;56:924-935.

Bishop NA, Lu T, Yankner BA. Neural mechanisms of ageing and cognitive decline. Nature. 2010;464:529-535.

Haigis, MC, Guarente LP. Mammalian sirtuins—emerging roles in physiology, aging and calorie restriction. Genes Dev. 2006;20:2913-2921.

Welberg L. A long and lean life. Nat Rev Neurosci. 2007;8:494-495.

The Neurobiology of Conscious Intent

Perhaps the seminal component of any clinician’s behavioral repertoire is the ability to understand the conscious motivations and intentions of their clients. This article addresses the work of conscious motivations at the neuroanatomical level.

I seldom address the notion of consciousness—let alone motivations—in this column for a very good reason. Nobody really knows what they are or even if there is a “they.” The literature is confusing, but it hasn’t stopped researchers from speculating on possible neuroanatomical and biochemical substrates that undergird the phenomena. Without a broad consensus about what is being studied, there can be no neurons, let alone molecules, for active experimental consideration. After all these years, researchers have yet to isolate an area of the brain solely devoted to the experience of consciousness. There may be none.

Given the importance of these issues to the mental health professions, I revisit the concept of motivations from time to time—but only when the data are conservatively presented, with sober, modest conclusions. The findings described here originate from experiments that have attempted to determine how we voluntarily choose to perform a motor task (action planning). This work requires reviewing background information on association cortices and the neural substrates behind a decision to initiate voluntary action.

Association cortices

Functionally, the cortical regions of the brain and their myriad interlocking circuits can be divided into 3 modules. These consist of front-, back-, and middle-end domains.

• Front-end functional domains are sensory information processing centers. The brain receives input from the eyes, ears, and other sensory systems. It sends the input off to various places for further processing.

• Back-end functional domains involve motor control systems. These systems essentially respond to whatever command the sensory cortices give to it (eg, execute a decision to move).

• The middle-end suite involves nearly everything other than front-end and back-end functional domains. These association cortices generally entail higher processing features and are some of the least understood and the most mysterious parts of the brain.

One such cortex, located in the inferior posterior parietal cortex, is a sensorimotor association region that links sensory stimuli to motor movement. It may even be involved in sensory prediction, which calculates the consequences of a given action through the simultaneous evaluation of input from both sensory (front-end) and motor (back-end) functional domains.

Volitional motor movement

Many of the actions humans initiate on a day-to-day basis seem to depend on a kind of internal free will. This sequence of events (also known as volitional motor movement) gives humans a sense of control: we act because we want to act. That is why researchers use volitional motor movements in their research designs. Researchers interested in volitional behavior study neural prime movers behind decision making.

Exactly what does it mean to want to do something? We do not really know. The events that initiate movement occur in a fairly straightforward sequence (although it depends on the source of the signal). For example, a central processing area with directives for voluntary motor movements pass through a final staging area before the execution of an action. This region is the primary motor cortex.

Research on laboratory animals demonstrates that this cortex decides on a course of action that depends on the source of signals it receives before the execution of that action. One source originates in the premotor cortex. Signals in this area initiate movements in response to a specific external trigger, such as a visual cue.

The second source arises in the presupplementary motor area, which is stimulated when laboratory animals make the same movements mentioned above, but they do not originate from responses to an external source. The movement instead arises spontaneously; a thought is internally generated through intentional actions. There is an observed rapid rise in electrical signals that build up just before the brain executes these actions. This has led to the notion that the presupplementary motor area harbors some kind of readiness potential, a useful function in generating movement (Figure).

In terms of human behavior, complex human brains have many more research issues to solve than standard laboratory animal research can address. One potential confounder is conceptual. With research of this type, scientists often tell subjects to choose (or not to choose) from a variety of options. Is that voluntary? Hardly. This is like saying, “Okay, it’s time to have some voluntary volitional behavior now,” or like runners at a race who respond to the starting gun. Do volitional actions disappear in these experiments with human subjects? Are these subjects simply reacting to commands to respond, not to respond, or to respond however they want? To test volition, researchers should not control the input. Nevertheless the experimenter must, almost by definition.

Wilder Penfield revisited

Another complexity involves engineering. How does conscious intent to move an arm relate to the actual movement of the arm? This could be partially resolved with electrical stimulation mapping in which surgeons create a map of the brain on conscious patients to understand what tissues need to be avoided during certain manipulations (such as resection). No pain neurons exist in the brain. The patient, immobilized in a stereotactic frame, can be consciously interrogated while the surgery takes place. The surgeon applies a gentle electrical current to the open tissue, talks to the patient about what he or she is experiencing, and makes a map that discerns what areas to avoid during cutting. Working primarily with epileptic patients, the legendary Canadian physician Wilder Penfield first performed these techniques.¹

This technique has proved to be of great value in understanding volitional components of motor movement. It was discovered almost 2 decades ago that if one stimulates a specific area of the human presupplemental area, the patient will experience a conscious urge to move.² This gets around the runner’s starting gun problem mentioned previously. An external electrical stimulator supplies a specific quantity of electricity—and a desire to do something is suddenly generated!

As important and well-characterized as these data are, they hardly explain what causes the presupplemental area to generate the signal in subjects not undergoing surgery. Some research findings answer this question and have led to some intriguing results.^3,4

When the inferior posterior parietal cortex was stimulated, the patient experienced an urge to move specific body parts. Stimulating one area caused patients to want to move their arms. Another region, the lips. Another region, the chest. This is similar to what one observes in frontal lobes, except that you are nowhere near the frontal lobes. Recall that this is the associative cortex region (a sensorimotor associative area at that), quite distinct from anything observed in the well-characterized general motor areas of the frontal lobes. Was this simply a remote stimulation?

This result showed that the answer would be no. The parietal cortex urges were qualitatively different from those obtained by stimulating parts of the presupplementary cortex. It is well known that if the presupplementary cortex is stimulated at a low current, the urge to act is acquired. However, if the same region is stimulated at high current, actual movement occurs. That’s not what happened in the parietal cortex. The urge was stimulated at low intensities, but movement was never generated at higher ones. Instead, subjects felt that they had already performed some movement.

This is important. The desire to move did not result from subtle motor contractions that may have been generated by motor regions (an alternative idea that has been put forth as a rational explanation for the results in previous experiments). Parietal stimulation never produces muscle activity, regardless of the intensity. The stimulation of the premotor cortex itself produces large-limb movements in subjects, but never the desire to move the limbs. They usually remain unaware that movement has occurred when these regions are stimulated.

These results suggest the presence of 2 specific aspects of conscious intention (however one defines it). One might be the conscious correlation of preparatory motor commands in the presupplemental cortex region, as is clearly observed in laboratory studies of animals. The other might involve sensory prediction of the consequences of those commands, under the domain of the association cortex region. A portion of conscious intent seems to be a specific class of experiences housed within the parietal lobe.

Conclusions

It appears that the parietal lobe contributes to the conscious experience of intention, at least in regard to motor movement. These results cement 1 more brick onto the great construction project that seeks to define intention. But they hardly hint at the overall building.

Pushing the edge of our understanding into the murky world of association cortex only means that future experiments will be trickier to interpret. Electrical stimulation mapping, as good as it is, is necessarily a blunt instrument that stimulates thousands of neurons simultaneously. Not isolated modules, these regions connect to each other in complex, little-understood ways. That the regions produce different behaviors is an important finding but not a defining one.

How do the frontal and motor aspects of volitional experience differ from the parietal, sensory versions? What factors stimulate the parietal lobes in the first place? What about remote effects?

Questions such as these remain to be answered and are just a few of the many that researchers will face as they attempt to define intentional and conscious experiences.

This article originally appeared in the Psychiatric Times.

References

1. Penfield W, Erickson TC. Epilepsy and cerebral localization: a study of the mechanism, treatment and prevention of epileptic seizures (Review). South Med J. 1942;35:222.
2. Fried I, Katz A, McCarthy G, et al. Functional organization of human supplementary motor cortex studied by electrical stimulation. J Neurosci. 1991;11:3656-3666.
3. Haggard P. Human volition: towards a neuroscience of will. Nat Rev Neurosci. 2008;9:934-946.
4. Custers R, Aarts H. The unconscious will: how the pursuit of goals operates outside of conscious awareness. Science. 2010;329:47-50.

Memory Reconsolidation and What Albert Ellis Knew All Along

I can almost hear Albert Ellis saying “Amen” to the data I am about to share. To explain his reaction, I have to talk about war.

As a former military brat (my father was an Air Force “lifer”), I grew up hearing about large-scale military plans designed mostly to contain the formidable Soviet threat. Once that threat dissolved, those large-scale strategies had to undergo a radical revision—an amending process that is ongoing today. We are learning to confront smaller threats in countries whose formal militaries we can overthrow in weeks, sometimes days, but whose informal warriors can take years to defeat. It is sometimes called “asymmetric warfare”: the operations in Iraq and Afghanistan are prime examples.

Many things concern me about our military operations in those theaters, but one of them touches on a professional interest—the growing incidence of posttraumatic stress disorder (PTSD). There are 3 reasons that prick up my research instincts, all springing from this asymmetric warfare.

• First, there are no real “fronts” to many of these operations. As a result, soldiers who used to be able to sit comfortably in the rear (often called the “tail”) are now as exposed to the hazards of combat as those charged with fighting (often called the “tooth”). Consequently, the pool of soldiers who are potential candidates for PTSD has increased. This means the research cohort is getting larger and increasingly better defined.

• Second, soldiers are returning into these asymmetric environments multiple times for very long periods. This provides an opportunity to study the mental health effects of combat over a sustained period of years—valuable for countries such as Somalia and parts of Uganda, which have been in a continuous state of war for decades.

• Third, the number of women exposed to combat is increasing. Since there is growing evidence that men and women process stress very differently, these populations represent valuable research opportunities to ask and answer sex-based questions regarding the effects of combat on behavioral outcomes.

All of these issues present mental health challenges, enormous scientific opportunities, and a compelling reason to vastly increase funding for research into the long-term effects of disorders such as PTSD.

Into this breech comes a very interesting finding of potentially great value for the mental health community in general and the American military in particular. It involves new insights into how the brain copes with fearful stimuli in the midst of a cognitive process called “reconsolidation.” The finding suggests powerful noninvasive behavioral protocols that might have real applications in the modern battlefield, and in this column, we are going to discuss it. We’ll start with some background information (dusting off some old skinnerian nomenclature), talk about the cognitive neuroscience behind reconsolidation, then move directly to the data.

PAVLOV’S BELLS

It might take removing a few cobwebs for you to recall your Behaviorism 101 lectures, particularly regarding “classical conditioning.” The CliffsNotes version involves pairing some neutral stimulus with an unconditioned stimulus (UCS). This pairing ends up eliciting an unconditioned response (UCR). Through a series of repeated pairing experiences, the neutral stimulus is transformed into a conditioned stimulus (CS). The CS then evinces a conditioned response (CR), which is similar to the UCR.

Does that ring a bell? It might, if we add back Pavlov and his canonical experiments involving teaching a dog to salivate whenever the animal hears one. The sound of the bell is the neutral stimulus as the experiment begins, which fully exploits the fact that dogs naturally salivate in the presence of food—something you don’t have to teach them. Food is thus the UCS, and salivation is the UCR. In a series of learning trials, the bell and the food are paired together. Whenever the animal hears a bell, the dog is presented with food. After multiple exposures, the dog salivates just to the bell. The bell is now transformed into the CS, and the salivation in response is the CR.

CONSOLIDATION

Understanding the concept of “consolidation” is considerably harder. There are many different types of memories, and different categories follow different consolidating rules. We are capable of experiencing classical conditions—combat veterans learn to pair very quickly the presence of tiny wires in dirt with a panic response. But they are also capable of remembering that the Battle of Hastings occurred in 1066, and most still know how to ride a bike. Different brain mechanisms are used for each type of memory, categories that are called by different names. The history lesson is termed “declarative memory,” for example.

When declarative memories are first formed, the traces are fragile, labile, and at great risk for extinction. To make a memory more durable, it has to undergo a series of processing steps (consolidation). Eventually the memories will reach a state in which they are infinitely retrievable and not subject to amendment. But it takes a great deal of time to render them permanent.

There is mounting evidence that every time you retrieve a memory—even ones you have fully consolidated—you return that memory to its former fragile, labile state. It then has to undergo another round of consolidation to stabilize—via reconsolidation. At the point where it is still fragile, it is temporarily subject to amendment, even loss. (The window is about an hour.) Although the particulars remain controversial, there is growing evidence that this Pause button exists in both animals and humans. It is this hour-long pause in the consolidation stream that a group of researchers in the psych department at New York University (NYU) used to do the following experiments.¹

THE DATA

The idea for the NYU experiment1 was very simple: suppose a person learned a fear response in a classical condition paradigm, then was asked to recall the experience at a later time into memory. What if, during the hour the reconsolidation Pause button is in the On position, and the subject was treated to new, non-threatening, non–anxiety-inducing information?

The researchers decided to find out. They used human subjects, mild electrical shocks delivered to the wrist, and squares of different colors projected onto a video screen as their materials. The level of fear was assayed continuously using standard skin conductance measures. Participants’ memory behaviors were measured 10 minutes after exposure to certain stimuli, 6 hours after exposure, and 12 months after exposure.

The first manipulation was divided into 3 parts, which can be divided into the days in which the actual research took place.

Day 1

Subjects were presented with a video screen that displayed squares of 2 different colors, which, as you’ll see, becomes the CS. Whenever subjects saw 1 of the colors (which I’ll call the “hot” color), they got a shock to the wrist. After a few repeated pairings, whenever the subjects saw the hot-colored square, their fear responses jumped considerably. The subjects were then divided into 3 groups, whose experiences I will talk about in a minute.

Day 2

The next day, all groups underwent repeated exposure to the same 2 colored squares—this time without the electrical shocks. The attempt was to introduce an extinction experience process, to defang the effects of the hot color—and it worked. After a while, fear was reduced even for the hot color—and in all 3 groups. But what happened before this delightful fear reduction separated the subjects into their respective groups?

The first group simply went through the extinction process as described above, functioning as controls. The second group was reexposed to the hot color (just once) without receiving the shock 10 minutes before the extinction process began. This represents the “new” information. The third group was also reexposed to their hot color (just once, and also without the shock) 6 hours before the extinction process began. This represented the same “new” information.

Why the difference in the period between the second and third groups? The idea for the second group was to bring back a fearful memory—but with new information about it—within their Pause button reconsolidation window (that’s why the 10 minutes) before undergoing extinction. The hot color was supposed to shock them, but it didn’t (which is what was new). So the fearful memory was retrieved but was soon greeted with this new, delightful finding, which would be reinforced when they underwent the formal extinction process.

The idea for the third group was to bring back a memory after the Pause button reconsolidation window (thus the 6 hours) before it underwent extinction. They, too, got delightful news, but there was no reinforcement within the 1-hour window. They had to wait 6 hours to start the confirming extinction procedure.

Day 3

The last day—which was 24 hours after extinction—all groups underwent the same shock regimens done initially. This was an attempt to reestablish the fear response to the hot color. How did the 3 groups respond? (See Figure.)

The first group—the controls—recovered their fear responses spontaneously. When they saw the hot color, their skin conductance immediately showed “fear.” The extinction procedures they had undergone the previous day were essentially a waste of time.

The third group, the ones who had been presented with “good news” but had to wait 6 hours before it could be reinforced, might as well have gone home. They showed the same spontaneous recovery and the same resistance to extinction.

The real research gold is what happened to the second group—the ones who had gotten “Hey, its going to be alright” information 10 minutes before extinction reinforcement procedures began. They showed no fear response when they were reexposed to the hot color. Their fears had been blocked by the administration of updated information. Even when tested a year later, the fear-response memory was still blocked. The effect was long-lasting, powerful, and event-specific. Controls were performed to ensure against a generalized bleed-through effect (this was to ascertain that interfering with the reconsolidation of 1 memory trace did not influence the processing of a similar stimulus).

CONCLUSION

This is a remarkable achievement. The ability to block fearful memories was done by capturing a reconsolidation window, without any pharmacological agent, but with effects as long-lasting as any medication. Memories marinated in apprehension could be blocked simply by updating them with positive information within a specific period of time. By amending the experience before the consolidating stopwatch struck “60 minutes,” a new perception, quite resistant to change, was obtained.

The potential mental health applications of this work are extraordinary, both for civilian and military populations. It may be very possible to boost the power of introducing non–self-defeating thoughts by exploiting the reconsolidation window. This would allow one to rewrite—even efface—toxic emotional memories, which would represent a noninvasive way of helping a person control his or her psychological interiors. In the future, an entire therapy may evolve around just such timing issues. These data are just begging for application-oriented investigations to commence.

Hear that rustling? That’s the sound of Albert Ellis, from his grave, mumbling his “Amen,” and possibly, “You see? I told you so.”

This column appears in the May 2010 issue of Psychiatric Times.

The Epigenetics of Stress

News: Brain Rules 2010 Workshops in Seattle, Denver, and Boise

Overly sensitive, aversive reactions to stress seem to run in families. The literature abounds with reports of relatives in these populations predisposed to depression, anxiety, and even suicide. Some family members present with glucocorticoid levels notched abnormally high, and in curiously deregulated concentrations. Behaviorally, they seem to exist at a permanent state of high alert.

Attempts to isolate the genetic underpinnings of this obvious hyperaroused stress sensitivity have met with mixed success. People carrying certain mutations in the serotonin transporter gene seem particularly vulnerable to the normal stresses and strains in life, although there have been difficulties in replicating all the findings. Plenty of people exist who are just as vulnerable to stress but who do not carry this mutation—or any other suspect genetic anomaly—that could explain the behavior.

It is now possible to characterize some of this seeming heritability—and accompanying statistical turbulence—without invoking heritability at all. This is the world of epigenetic transfer, the ability to pass on a trait without having to stop at a meiotic border. A recent study has demonstrated how a powerful environmental stressor can exert molecular effects that last over a long period. The mechanism is epigenetic. It is the first result to characterize a molecular mechanism, induced by early life stressors, that influences behaviors penetrating into adulthood.

To explain these findings, I will first talk about transplacental cortisol and its effects on the developing fetal brain. Then I will move to the data, which do not involve humans at all.

The starting observation

For years, we have known that stressed wombs tend to produce stressed babies. Most molecular explanations for this observation invoke the effects of transplacental glucocorticoids on fetal brain development. If mothers become too stressed (so the idea posits), too many stress hormones enter the womb, penetrate the fetal brain, and interfere with its proper development.

There is some empirical support for this notion. First, excess levels of maternal glucocorticoids have been shown to cross the placenta, targeting the fetal limbic system and causing it to develop much more slowly than in typical controls. This is thought to result in future behavioral dysfunction, particularly regarding reactions to external stressors. It specifically hampers the developing “braking system” of the hypothalamic-pituitary-adrenal (HPA) axis. (As you recall, the HPA axis is a series of biochemical reactions to threat that involve interactions between the hypothalamic, pituitary, and adrenal glands.)

Social states experienced in early life can directly affect later behavior. Until now, exactly what molecular mechanisms undergird such effects have remained a complete mystery.

This embryonic braking system is a coordinated series of responses that normally result in the inhibition of glucocorticoid production after some environmental stress has been successfully negotiated. Without this braking system, the fetal brain is wired to produce excess glucocorticoids in an increasingly unregulated fashion. The baby carries this deregulated system into adulthood. If the adult is female and becomes pregnant, her system, which is already flooded with cortisol, marinates her new baby with glucocorticoid. This once again creates a hyperaroused womb, complete with new fetal damage. The trait is thus passed along, not through the germ line but simply through womb exposure.

Although a great deal of work needs to be done to complete this admittedly depressing story, large parts have empirical support. This includes some intimate, biochemical details. Recently, a molecular mechanism has been uncovered that may explain this nongenetic inheritance. Although work has been done mostly in rats, there are broad implications for human behaviors. After a brief explanation of an epigenetic mechanism involving DNA methylation, it will be to these data that we turn next.

Early life stress

The work to be described involved inducing behavioral stress in a cohort of laboratory rodent pups, and then watching the effects of that stress on behavior as the rats matured. The standard protocol to induce developmental behavioral stress is to apply an infant-mother separation schedule early in postnatal life. Typically, the animal is separated from its mother 3 hours a day for the first 10 days of its life. The experience of early life stress (ELS) results in a lifelong elevation of glucocorticoid secretion and a disruption in normal stress responses (a permanent and heightened endocrine reaction to externally supplied stress). The animal becomes hyperaroused, presenting an abnormal regulation of the HPA axis. This arousal induces broad behaviors associated with mood and cognitive disorders.

A number of important hormones regulate the HPA axis, including 2 hypothalamic secretagogues—corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). When a stressful experience is encountered, signals arise that increase the synthesis and release of pituitary CRH and AVP. There is a rich history linking CRH and AVP to cognitive and affective disorders; their receptors are the targets of a number of psychopharmacological medications. AVP is also expressed in a specific subset of neurons within the hypothalamic paraventricular nucleus.

Understanding the molecular mechanisms behind the regulation of the HPA axis involves, in part, understanding how the AVP gene is regulated. Like all genes, AVP has a promoter region (which, as you recall from your undergraduate biochem class, functions as an on/off switch for the gene). A number of regulatory sequences surround this on/off switch, controlling its transcriptional activity. What happens to these sequences ultimately determines the role AVP plays in regulating stress responses (Figure).

One biochemical modification of the AVP promoter involves a process of methylation, the replacement of a hydrogen atom in certain nucleotide residues (generally cytosine residues) with a methyl group. This process is mediated by a class of enzymes termed “methyltransferases.” Many sites along the chromosomes are naturally methylated, but the process is in equilibrium, and many residues don’t stay that way. The amount of methylation on the regulatory region of a gene can control its transcriptional activity.

Methyltransferases are enzymes whose activities are also tightly controlled. One protein involved in regulating their activity is called MeCP2. This protein is specifically involved in recruiting proteins that will assist in the methylation of DNA. If MeCP2 activity is inhibited, the methylation of specific regions of DNA will also be inhibited, and the region is said to be hypomethylated. Because methylation affects a promoter’s ability to regulate the gene to which it is attached, one way to control gene activity is to control MeCP2 activity.

Results

With this background information in mind, we are ready to talk about the data. A typical ELS protocol was instituted immediately after the rats were born. Biochemical assessments began when the animals were 6 weeks old and continued for the next 10 months. A total of 4 findings emerged from these studies:

• ELS animals secreted abnormally high levels of stress hormone (specifically corticosterone) when compared with control groups. This excess persisted over the life of the experiment.

• ELS animals experienced a sustained rise in vasopressin expression (messenger RNA) in the hypothalamic paraventricular nucleus. That’s significant. This region of the hypothalamus is involved in regulating hormones associated with stress. Reassuringly, this elevation could be reversed with the application of an AVP receptor agonist.

• There was reduced methylation in a specific regulatory region for the AVP gene.

• The change in methylation was shown to be a result of the inactivation of MeCP2.

The behavioral changes in these mature animals were what you would expect in hyperaroused humans. They were less able to cope with stress in a wide variety of testing conditions, and they showed persistent memory impairments when compared with control groups.

Conclusions

These findings are quite stunning. For the first time, a molecular change associated with a persistent hyperaroused state in an adult animal was induced by behavioral neglect during the animal’s extreme youth. It is axiomatic from a counseling perspective that social states experienced in early life can directly affect later behavior. Until now, exactly what molecular mechanisms undergird such effects have remained a complete mystery. These data reveal strong epigenetic factors.

The normal caveats apply, of course. The experiments were done in laboratory animals, not humans—a red flag for anyone trying to apply animal-based behavioral results to human patients. Also, the stress was experienced after the pup was born, not during gestation. No measurements of transplacental glucocorticoid trafficking were made in these experiments. Nor was the post-ELS next generation examined. The behaviors induced in the offspring of these animals is an area of future research.

None of these objections hurt the findings, of course—just their interpretation. Their real value lies in what they portend for the future. Uncovering a mechanism induced in childhood that mediates a persistent behavioral experience in adults is a phenomenal achievement. Showing that these associations sometimes have epigenetic underpinnings is an added bonus. These results serve as flashlights, directing where scientists interested in human reactions should spend their next research dollars. Stress responses really do run in families. Showing that some reasons for this may be mostly environmental—with no DNA in sight—is the biggest plus of all.

This article appeared in the April 2010 issue of the Psychiatric Times.

The Genetics of Temperament

There are a lot of temperamental Jerome Kagan moments in my friend’s household—an observation that will require this entire column to explain. What exactly is a temperamental moment? And who exactly is Jerome Kagan?

Before we start, it might be useful to know that my friend’s household consists of a husband, a wife, and 2 little girls, aged 6 and 9. Their temperaments could not be more different. The 6-year-old is little Miss Sunshine—socially fearless, prone to risk taking, ebullient, confident. She will charge into a playroom full of strangers, initiate 2 conversations at once, quickly survey all the toys in the room, and then lock down on the dolls, playing with them for hours.

Her big sister behaves in exactly the opposite way. She seems fearful, tentative, cautiously tiptoeing into the same playroom after only reluctantly leaving her mother’s side. She will then find some safe corner and just sit there. It is a bit sad to watch; this older girl seems to show no interest in exploring, hardly speaks at all, and appears scared if somebody tries to talk to her. She is extremely averse to taking risks of any kind.

The younger daughter doesn’t have a shy bone in her body. The older daughter appears highly reactive, anxious, and afraid. Yet, they both come from the same family. What is the difference between these 2 kids?

Psych 101

A partial answer comes from the laboratory of legendary psychologist Jerome Kagan, in case you’ve forgotten your Psych 101. Many years ago, Kagan noticed that babies seemed to fall into a reactivity continuum. Some kids at one end of the spectrum behaved like my friend’s self-confident 6-year-old girl. At the other end resided kids who were like the self-doubting 9-year-old. And there were kids whose behaviors fell between the extremes.

Temperament provides the emotional and behavioral building blocks on which adult personalities are constructed.

Kagan was the first to posit that where a child sat on this continuum—a description of one dimension of their temperament—was in large part hardwired—or at least, observable shortly after birth and mightily resistant to change. These traits may even be genetic. Now that several longitudinal studies have matured and their research paybacks have long since been cashed, Kagan’s insights have been shown to be well-placed. Temperament is resistant to change, depending on how you define it, and there may even be a few genes that help explain the behaviors. Because it may have been a while since your last psych class, I thought you’d like an update.

We will begin by briefly reviewing the definition of the notion of temperament, spend some time discussing Kagan’s initial experiments, then move to a genetic system that shows great promise in helping to describe the DNA behind the behavior. Feel free to skip to the section, “The gene work,” if Kagan’s great body of research is part of your vocabulary.

A review of temperament

In the cognitive neurosciences, temperament is a multilayered concept. It is often defined as a person’s characteristic way of responding emotionally and behaviorally to external events. These responses are fairly fixed, innate, and observable soon after birth and may have genetic components. Personality is defined as not being so immutable. It is shaped primarily by the parental and cultural factors into which a child is born and raised.

Personality is influenced by temperament, the same way a house is influenced by its foundation. Many researchers believe temperament provides the emotional and behavioral building blocks on which adult personalities are constructed.

There is some real controversy about the edges of these definitions, of course—conflicts typical of the “nature/nurture” battlefields researchers have been fighting for decades. Jerome Kagan was really the first to sink some empirical teeth into an argument previously based on opinion. He screened 400 kids way back in 1979, doing experiments in places similar to the rooms in which the 6- and 9-year-olds mentioned above were playing. He found about 15% acted like the younger daughter (behaviorally ebullient, or low-reactive [LR]) and another 15% acted like the older daughter (behaviorally inhibited, or highly reactive [HR]). He measured a subset 5 years later and found things were remarkably stable. Only 3% of the children had actually changed categories.

Kagan next investigated 500 kids starting at 4 months of age, coding them using the same LR or HR behavioral algorithms as before. He retested these same kids at 4, 7, 11, and 15 years (the experiment is still ongoing, although Kagan has retired and bequeathed the work to another colleague). He found that HR babies were 4 times more likely to be behaviorally inhibited by age 4 (like my friend’s older daughter). By age 7, some form of anxiety had developed in half of these kids, compared with 10% of the control HRs (who made up about 20% of the population).

As HR kids navigated through school, Kagan noticed most were academically successful, even if they were a bucket of nerves. They got good grades and made lots of friends. They were less likely to experiment with drugs, to get pregnant, or to drive recklessly—perhaps because of an anxiety-driven need to acquire compensatory mechanisms to socialize properly. Kagan actually liked HR people and regularly employed them during his research career. “I always look for high reactives,” he told The New York Times in 2009. “They’re compulsive, they don’t make errors, they’re careful when they’re coding data.”

Kagan’s conclusion? Babies come into this world with an inborn temperament. About 20% are born with a predisposition to anxiety—a behavioral type that remains remarkably stable over time. Kagan calls this the long shadow of temperament.

There are, of course, confounders and nuances to these conclusions, familiar to anyone who does behavioral work for a living. Kagan and colleagues have shown that these findings are not necessarily deterministic. Not every brain state sparks the same behavior. Nor do they say everything there is to know about temperament. Kagan freely admits he is only studying 1 dimension of the phenomenon—the reaction to new things. At least 6 different dimension scales have been cited by researchers in the field at various times to describe this multidimensional idea, ranging from fearful distress to attention span/persistence to rhythmicity. (For the complete list, see the Figure.)

Most important, these studies make conclusions about tendencies, not destinies. Even when the trends are strong, the predictive power of Kagan’s work runs in 1 direction. The data do not forecast what these children will become as much as they predict what they will not become. HR infants will not grow up to be exuberant, outgoing, bubbly, or bold. The older daughter will never become the younger daughter.

Even with these caveats in mind, the data almost beg for genetic research. The fruit of one of these inquiries is described next.

The gene work

The discoveries suggesting genetic components to temperament started with that workhorse of the human behavioral lab—twin studies, monozygotes versus dizygotes versus siblings versus strangers. Clear findings emerged. Using infant temperamental inventories, the degree of resemblance (correlation) for identical twins was shown to be about 0.4. For fraternal twins and non-twin siblings, the resemblance score was between 0.15 and 0.18. In these studies, about 60% of kids who seemed very inhibited as youngsters grew up to be inhibited as adults. They were prone to the same strong fears and phobias shown previously and were the most anxious in the group. Ninety percent of the uninhibited group stayed uninhibited as a group when adults.

A 0.4 resemblance score is enough to start thinking about DNA. Not surprisingly, molecular biologists have gotten into the fray in the past few years. It has admittedly been tough going. The twin findings hardly represent a 100% determinative slam dunk, and that’s because of this work’s ancient foe—nature versus nurture. Discovering what percentage of nature and nurture exists within any human behavior is hard to do, plagues even the most robust research of this type, and is not accomplished in the gene I am about to describe. The word “partial influence” or “susceptibility” could—and should—be used for the following.

There is a gene that is considered by some researchers to play a large role in the ebullient risk takers at the far end of Kagan’s findings. It is sometimes called the risk-taking gene, dopamine receptor D_⁴ (DRD4). This protein is a member in good standing of the range of molecules capable of binding to dopamine and exerting its specific physiological effects.

There is a large number of variants (polymorphisms) in this gene. One that has received a great deal of attention is a variable number terminal repeat (VNTR) in exon 3. That last sentence is a bit dense and is described below.

As you know, DNA is made up of long strings of nucleotides, As, Gs, Ts, and Cs. A VNTR is simply a region on the chromosome that has been organized into a tandemly repeated sequence. A tandem repeat is a repeated sequence of nucleotides that are jammed together. They can be quite long. Because VNTRs are on a chromosome, they are inherited.

An exon refers to the structure of a gene as it lies on a chromosome. As you may recall, structural genes in eukaryotic cells are discontinuous. Genes are sprinkled with regions that code for protein (called exons) and regions that code for nothing (called introns). They can possess VNTRs. Exon 3 is simply the third exon from the starting sequence and possesses the VNTRs in question. Each VNTR is 48 base pairs in length.

DNA samples from many people have been isolated, and the DRD4 gene characterized. They are infested with 48–base pair VNTRs, which can be divided roughly into “short form” and “long form” versions of the repeat. The short form is considered to be under 6 repeats of the VNTR. The long form is anything over 6 tandemly repeated sequences.

The collision between the DRD4 polymorphisms and Kagan’s work occurred more than a decade ago when it was noticed that people who scored high for risk-taking behaviors possessed the long variant of the DRD4 gene. Those who did not, carried the short form. This association has been replicated several times.

Further studies with these polymorphisms seemed to show a parenting effect. Most kids who have an emotionally distant parent develop “externalizing behaviors,” heart-breaking attempts to get their parents to pay attention to them. Those behaviors vanish if the kid has a specific variant of the DRD4 gene. They seem Teflon-coated, stress-resistant, and show little probability of becoming emotionally insecure.

Of course, all these data are correlative—the causal work has yet to be done—which means there’s a lot more work to do. One central mystery is figuring out exactly how low- or high-reacting behaviors and risk-taking behaviors relate to each other. DRD4 does lots of things besides mediate temperament. The role of other genes has not yet been determined. Most behaviors of this complexity are the result of dozens of interacting gene sequences.

So, the work continues. It is always marvelous to see well-done behavioral research starting to be fleshed out in molecular terms—even if there is a long way to go. The information certainly seemed to soothe my friends when I told them about it. They, like all good parents, felt the behavioral differences in their little girls were entirely a result of their failings as parents. In fact, the daughters may have been guilty of nothing more than, for better or worse, choosing their parents the way they did.

This article appears in the March issue of the Psychiatric Times.

Crime, the Hippocampus—and the Lingering Eye

Let’s say you’re in a crowded bar when somebody suddenly shoots at a patron. You clearly see a man carrying a firearm, but all hell breaks loose as you and everybody else scramble for the exits. In the terrifying seconds following the crime, you lose track of who discharged the firearm: it could have been 1 of 3 suspects. Afterward, the police interview you, but it is hopeless. Even bringing in the suspects for a lineup isn’t going to help you recall. There will be no “Perry Mason” moments, when the perpetrator breaks down under the weight of guilt and confesses to the crime. How can the authorities make an arrest?

They will be in for a tough time. Law enforcement officials know better than anybody about the long and storied literature concerning false memories, and the extraordinary unreliability of eyewitness accounts. People make things up all the time in high-pressure situations—seeing things that weren’t there, omitting things that were. The brain obviously records the information as it occurs in real time. But accuracy becomes only one version when the brain makes a conscious effort to retrieve the events.

What if there were a technological way to distinguish between these 2 versions of the same experience . . . the event that actually happened, and the version you perceived to have happened? The topic of this month’s column is an amazing result that promises to make this distinction possible. Using a combination of eye-tracking devices and noninvasive imaging, a group of researchers have uncovered an interesting way to get at a more accurate version of an event, even when conscious retrieval breaks down.

To talk about this extraordinary discovery, we will first need to review a few processes regarding human memory and some of the neural substrates that undergird it. Feel free to migrate to the “The results” section if words like “explicit memory” and “relational memory” processing are working parts of your vocabulary.

Thinking about memory

Present-day researchers use many ways to categorize human memory, some of which are clearly contradictory. That’s because there are many types of memory systems and subsystems, and not everybody agrees on just what they are. Is learning to ride a bike different from learning a foreign language? If a memory involves an emotionally competent stimulus, is that memory qualitatively different from the memory of a boring list of dead monarchs in a history class?

To make matters more complicated, many of these memory systems work in a semi-independent fashion. From a research perspective, the only clear thing you can say is that memory is not yet a unitary phenomenon.

One popular way to classify human memory abilities is based on whether the memory requires conscious awareness for retrieval. This idea was eventually transformed into a classification system involving so-called declarative memories and non-declarative memories. Declarative memories involve information you can physically declare—for example, that Thomas Jefferson was the third president of the United States. Non-declarative memories involve information you can’t declare—such as the learned ability to ski. Declarative memories are often called “explicit” memories; non-declarative memories are often called “implicit” memories.

The reason this classification system is so convenient is that there are distinct neural mechanisms and subsystems that underlie their function. Declarative memories are considered to be the province of the crown jewel of the medial temporal lobe—the hippocampus. The organ is ground-zero for helping to convert short-term declarative memory traces into long-term information. Damage to the hippocampus can lead to a selective inability to retrieve conscious events and facts while leaving non-declarative memory traces virtually intact.

Of course not everyone agrees with this rubric—the view is necessarily simplistic and has had recent empirical challenges. Certain patients with hippocampal damage possess deficits that are also associated with implicit memory formation. Some researchers believe the hippocampus is involved in yet another hypothesized memory category, so-called relational memory. This is the memory for the associations between discrete elements in an individual experience. Research findings in some labs implicate the hippocampus in establishing this type of memory too, whether a subject is aware of the experience or not.

It is also clear that other regions support the hippocampus in its quest to mediate memory formation, regardless of the category chosen. The prefrontal cortex (PFC), which is involved in decision making, planning, and foresight, clearly has a say in what will be encoded, stored, and ultimately retrieved. It communicates with the hippocampus during memory formation.

So what are we going to do? Even given these complexities, clear progress has been made in discerning the relationship between awareness and memory. Though it doesn’t settle the definitional issues, the data presented here show real promise of being practically helpful in a wide variety of settings. There may even be some real practical benefit to the law enforcement community.

The results

The inaugural idea of the project about to be discussed here was to explore whether the hippocampus could actually support indirect expressions of memories when conscious retrieval was failing. There were 2 machines involved in the experimental protocol. The first was a functional MRI, with special focus on images derived from the PFC and the hippocampus. The second was a tracking device that could detect eye movements and could determine where—and for how long—experimental subjects lingered over a given object in their visual fields. The eye-tracking device measured what commentators call REMEs—relational eye movement effects.

With these 2 machines calibrated and ready to work, the following 3-step experiment was deployed:

1. Healthy volunteers were shown pictures of individual faces superimposed on individual scenes (outdoor landscapes). The experiment was reminiscent of how animators draw characters on transparencies (cels), and then place the cels on previously painted backgrounds when recording for film.

2. After a delay, these volunteers were then presented with one of the previously encountered background scenes—sans face—and were asked to recall which face had been previously superimposed on it.

3. Finally, subjects were presented with a choice of 3 faces, and asked to choose which face actually went with a particular background scene in question. There was only 1 correct answer (2 of the 3 faces were distractors).

At each point, the subjects’ eye movements were tracked at the same time their brains were being imaged in the critical 2 seconds after the completion of the conscious retrieval task. The researchers were looking for:

• What the hippocampus did un-der conditions in which subjects matched the correct face with the correct background.

• What the hippocampus did when the subjects didn’t correctly match the items.

• What the REMEs did under both conditions.

What they found was extraordinary (Figure).

Extraordinary findings

Here are the results obtained when subjects were presented with the choice of 3 faces and correctly identified which face went with the background being studied:

• Activity in the hippocampus increased.

• Activity between the hippocampus and the PFC also increased.

• The eyes spent the most time lingering on the correct face (REME scores increased). There was a delay of 500 to 700 milliseconds between hippocampal activation and eye movement.

Here’s what happened when the subjects were presented with the choice of 3 faces and incorrectly identified which face went with the background being studied:

• Activity in the hippocampus increased.

• Activity between the hippocampus and the PFC decreased.

• The eyes spent the most time lingering on the correct face—even though the subject consciously gave a wrong answer.

The REME scores still increased. The eyes appeared to “know” what the correct answer should be, even though the subject did not consciously return the correct answer. By “know,” I really mean that the neural regions were directing the eyes. At some level, the brain was aware of the correct answer all along. For whatever reason, the organ did not return the correct answer to the subject. It means that accurate memory retriev-al was evident in eye movement behavior even when the subject’s judgment was incorrect.

This is a bombshell of a finding. Even if the subject was unable to recall a learned relationship between the face and the background, it appeared that the eyes and the hippocampus retained some of the information. And the eyes still responded correctly. At the very least, these data demonstrate that something beyond the hippocampus is required to make a previously encountered memory conscious.

These results suggest other ways to measure hippocampal memory formation that don’t depend on the subject’s conscious reporting. What other brain regions might be needed? The clue may come from the differential communication with the PFC. The connections between cortex and hippocampus became far less lively when the subject made a mistake. Because the PFC is involved in decision-making processes, its recruitment is hardly a surprising find. The intriguing decrease was not expected, however. It suggests new experimental directions in attempting to understand what the brain goes through when mistakes are being made.

One has to be careful of data like these, of course. One factor not controlled for in these studies is whether the subjects were conscious of the correct selection at the same time their eyes lingered on the matching face. They may have been aware of the correct choice, but in the intervening brief time between seeing the picture and selecting a face, they second-guessed themselves.

That’s hardly a deal killer. The real contribution of this work is to show that there are other ways of retrieving accurate information than direct interrogation of the witness.

And that has some powerful implications, especially for law enforcement professionals. Hooked up to these machines, you as a witness may indeed be confronted with a lineup of suspects who may have fired the gun in the bar. Your conscious recollection is just as fuzzy as ever, and you report that you cannot positively ID anybody. Your eyes (unbeknown to you) are consistently drawn to the actual perpetrator, however, revealing the correct version of the event. The machine detects this and the perpetrator, in the face of overwhelming technological superiority, breaks down and confesses. Sound like a fantasy? In the future, that may only be true of the perpetrator’s reaction.

This article originally appeared in the February 2010 issue of the Psychiatric Times.

The Cellular and Molecular Substrates of Anorexia Nervosa, Part 2

Correction: I should have mentioned in my article “The cellular and molecular substrates of anorexia nervosa”, posted in the Psychiatric Times on (part 1) November 1 and (part 2) December 7 2009, that much of it was a digestion of an excellent review of the topic in Nature Reviews Neuroscience, 10, 573-584 (2009), by Walter H. Kaye, Julie L. Fudge and Martin Paulus.

I think I am going to talk about the neurobiology of happiness in my next column. The reason has to do with the nature of our 2-month journey into the biology of eating disorders—a subject that, considering the dearth of explanatory data, is tough to write about. It’s also a bit depressing, considering how difficult it can be to treat. This is the second installment in a 2-part series that focuses on the neurobiology of restricting-type anorexia nervosa (AN).

Last month, I discussed behavioral and cellular aspects of AN.¹ A testable hypothesis was outlined: AN was described as a conflict between an un-acquired biological need to have food and an acquired negative reaction to it. Patients with AN recruit cortical executive reactions in response to appetite cues, reactions that insert a top-down “food-negative” bias into the normal drives for fuel. These executive reactions are consistently overstimulated in AN patients, leading to high anticipatory behavior and obsessive concern with future events. Derived mostly from noninvasive imaging studies, this notion of conflicting priorities (complete with a dysfunctional reward/punishment system) has surprising empirical support.

But it is hardly the complete story of AN. Besides behavioral and cellular concerns, there are also molecular interactions to consider. It is to these efforts that we turn, focusing on the “usual regulatory suspects” of dopamine and serotonin neurotransmitter biology.

A reason for genes

Many twin studies have been initiated in the attempt to characterize potential underlying genetic components to AN. There has been some success, and in 2 directions. Large-scale studies have demonstrated that between 50% and 85% of the variance observed in AN (and bulimia) can be attributed to genetic factors. The numbers actually suggest a continuum of diffuse but related behaviors—including weight dissatisfaction, weight preoccupation, and dietary restraint.

The second direction takes into account child temperament issues. It has been known for years that specific personality traits observed in adolescence can predispose an individual to AN. These include perfectionism, harm avoidance, and certain obsessive-compulsive behaviors. Genetic studies show these traits to be heritable as well. These are independent of body weight and can be present in unaffected family members.

One is continually confronted with complexity, confounders, and nuance. Not a welcome comment regarding a disease such as anorexia nervosa, which has the highest mortality rate of any psychiatric disorder.

The aggregation of these 2 lines of work gave researchers ample reasons to seek underlying genetic causes for the disorder. They are still looking. Investigation of the obvious choices—dopaminergic and serotonergic systems—has yielded some fruit. But the picture that emerges is far from complete, and at this point gives only tantalizing hints about potential molecular mechanisms.

Dopaminergic interactions

Behavioral work suggested ample reasons to suspect dopamine-pleasure responses might be dysfunctional in AN patients. Afflicted individuals often seem addicted to exercise. They are ascetic, anhedonic, and find precious little in their lives that is consistently rewarding (aside from the pursuit of weight loss). This “trait” versus “state” issue is strengthened because such behavioral patterns persist, albeit in reduced form, after successful treatment. Dysfunction in dopamine regulation, especially in the striatal circuits mentioned last month, might provide an important component to alterations in these behaviors. They may also play a role in the motor functions and decreased food consumption behaviors typically associated with AN.

Four lines of evidence support an involvement of dopaminergic processes in at least some types of AN:

• Concentrations of dopamine metabolites in the cerebrospinal fluid (CSF) of both affected individuals and recovered individuals are lower than in those without AN.

• Patients who have AN often present with difficulties in certain visual discrimination learning tasks. This is not a trivial finding. Studies show that such impairment often reflects a malfunction in dopamine-signaling.

• There are dopamine receptor (DR) D2 gene polymorphisms associated with those suffering from AN. (DRD2 is one of a family of dopamine receptors in the human genome.) A polymorphism is an aberration in its normal gene structure. The more tightly the polymorphism is associated with a given behavior, the more likely it is to exert an influence on it.

• Noninvasive imaging studies—positron emission tomography (PET) scans, mostly—found a surprising restoration in dopamine receptor binding activity in recovered patients. (Previous work had shown deficits.) Successfully treated patients presented with a dramatic increase in DRD3 binding—another dopamine receptor—in the ventral striatum. As you recall, the ventral striatum helps regulate reward stimuli. Its function played a prominent role in the cellular explanation put forward in the previous column. It must be noted that these PET scans are interpreted as changes in activity, but that could mean many things. The signals could indicate increased DRD3 densities, decreased extracellular dopamine, or both, in recovered patients.

Serotonin interactions

Compelling as some of these data are, dopamine is not the only neurotransmitter under active investigation. Other evidence suggests that compared with healthy individuals, people who are susceptible to AN have alterations in normal serotonin biology.

Most of the best work has focused on 2 ideas (Figure). The first posits that people vulnerable to an eating disorder have increased extracellular concentrations of serotonin—something that can actually be measured. The second is the presence of an imbalance in postsynaptic serotonin receptor activity. As you will recall, serotonin receptors consist of a family of related proteins. Two of these—the 5-HT_1a and 5-HT_2a receptors—have received a great deal of research attention.

There is reason to investigate alterations in ligand/receptor binding with these receptor systems in patients who have AN, especially given the mean age at onset. Gonadal steroid changes associated the menstruation (and stress) have been known to alter the activity of the 5-HT system during adolescence. Starvation-induced reductions in levels of extracellular 5-HT, for example, might result in reduced stimulation of postsynaptic 5-HT_1a and 5-HT_2a receptors, leading to behavioral alteration. The resulting dysphoria, normal in unaffected individuals, might be exaggerated in patients with AN.

There are testable questions surrounding these ideas. Forcing AN patients to eat, for example, might stimulate postsynaptic 5-HT_1a and 5-HT_2a receptor activity. This stimulation would lead to an elevation in dysphoric mood, transforming eating and weight gain activities into traumatic stress-inducing experiences. This might explain the no-win behaviors so common in AN patients. If the patient were allowed to continue to starve herself, anorexigenic information related to neuropeptide alterations (reduced b-endorphins, elevation in stress-related metabolism such as elevated corticotropic-releasing hormone), might exacerbate AN symptoms by driving food-restricting behaviors. Whether eating or starving, the same dysfunctional circuitry would be stimulated, all leading to the symptoms.

Do any of these speculations have empirical support? Two lines of evidence suggest an important involvement with serotonin in certain types of AN. However, the specific answers await further research.

• There is specific evidence that patients with AN present with an imbalance in postsynaptic 5-HT_1a and 5-HT_2a receptor activities in specific areas of the brain. These alterations might contribute to the feelings of abnormal satiety and excessive harm-avoiding, anxiety-riddled behavior.

• Persons with AN show unique anxiety-related 5-HIAA metabolic perturbations. The weight loss in these patients results in a reduction in 5-HIAA CSF levels. But they concomitantly show dramatically elevated 5-HIAA receptor binding in specific cortical and limbic structures—something not seen in healthy controls. Food might very well be anxiogenic in these individuals.

These findings, real as they are, do not provide many solid hints about molecular explanations for AN. To date, no single biochemical alteration has been shown to be both necessary and sufficient to produce the disease. Combined with the dopamine work, one might be tempted to say diseases.

And, I suppose, that is the frustrating point. Whether one is looking at behavioral components, neural circuitry, or molecular interaction, one is continually confronted with complexity, confounders, and nuance. Not a welcome comment regarding a disease such as AN, which has the highest mortality rate of any psychiatric disorder. It is one of the most expensive to treat, too, with no guarantee of success when therapy reaches its end point. Progress has been made, but we have a long way to go before we know everything. Given its urgency, that’s a very depressing thing to write.

Like I said, I think in my next column I am going to talk about the neurobiology of happiness.