New Chemical Lows in Brain Surveillance

As a profoundly wrinkled, 3-lb mass with a consistency of thick custard, the brain poses a puzzle for anyone who wants to understand how this cantaloupe-sized organ can do so much.

"Each word, each meaning, each motor movement, each thought, each response has to be embodied in some state in the brain," says electrochemist David Sulzer of Columbia University. For the brain to achieve all that it does in a way that changes and adapts over a lifetime of learning and experience, he says, "it essentially has to have an infinite number of states." For that to be the case, Sulzer surmises, "you need to have some extraordinarily complicated sets of mechanisms."

Anatomists and physiologists have been uncovering some of these mechanisms for more than a century. With its 100 billion or so neurons-each one a living electrochemical wonder that connects via cell-to-cell synapses with tens, hundreds, or sometimes many thousands of other neurons-the brain's cellular architecture itself offers a gargantuan number of possible states for storing and processing information.

A recent calculation by Danish neuroscientists suggests that the human brain's neocortex alone harbors approximately 150 trillion synapses. Add to that architectural complexity the ability of neurons to rewire their synaptic liaisons, tweak their chemical microenvironments, and change their firing patterns, and the number of possible brain states skyrockets.

It doesn't stop there. "Almost everything we look at turns out to be more complicated than we thought," says Sulzer, who is among a cadre of researchers who use electrochemical techniques to observe the chemistry that unfolds around individual neurons. For his part, he, along with his colleagues, has used a technique known as carbon-fiber amperometry to reveal details about a newly recognized level of neural complexity. This level involves the way nanoscale vesicles in the synapses of neurons release their cargo of neurotransmitter molecules such as dopamine and serotonin.

"People have been doing electrophysiology [to study neural firing patterns] for a long time, but that always translates into chemical signals," observes chemist Robert T. Kennedy of the University of Michigan, Ann Arbor. He and his coworkers have used electrochemical probes to measure insulin release from individual pancreas cells and investigate how that release feeds back on the behavior of the cells, a dynamic akin to the way neurotransmitter release affects nearby cells. As Kennedy sees it, scientists like Sulzer now are beginning to obtain the data needed to discern the "spatial temporal mix of chemicals underlying brain activity."

The bare-bones picture of neuron-neuron interactions in the brain goes something like this. A presynaptic neuron, having received chemical inputs from other cells, is either triggered to fire or not. A firing neuron is one in which the inputs to it, which generally impinge upon the dendrites on the body-side of the cell where the nucleus is located, collectively trigger an "action potential." When that happens, a wave of ionic movement across the cell's membrane travels down and around the neuron's body along its axon. An axon can extend to adjacent neurons in the brain or as far as the bottom of the spinal cord. The ionic movement finally reaches the cell's axonal terminals, which form synapses with wispy, signal-receiving dendrites of the postsynaptic cell.

When the wave of depolarization reaches the synapses, another ion-initiated mechanism kicks in and results in the release of vesicle-housed neurotransmitter molecules into the tiny synaptic cleft between the presynaptic neuron and the postsynaptic neuron. If enough of these neurotransmitter molecules bind to enough receptors on the postsynaptic neuron, the latter can be triggered to initiate an action potential of its own.

The classic view of the final neurochemical step of neurotransmitter release, known as "all or none" exocytosis, holds that vesicles in the cytoplasm migrate to and fuse with the membrane of the presynaptic neuron and then dump their entire load of neurotransmitter molecules into the synaptic cleft. About 10 years ago, however, scientists began finding that the classic view was too simple.

Extremely sensitive amperometric techniques allow oxidizable molecules released from, say, a synaptic vesicle, to be measured and counted by means of a well-placed microelectrode. Using these techniques, researchers began finding evidence that some vesicles use a "kiss-and-run" form of exocytosis. In this mechanism, the vesicle briefly fuses with the cell membrane, spews out some portion of its neurotransmitter load into the synaptic cleft, and then recedes into the cell, where it can quickly and efficiently be reused or recycled.

In their measurements of cultured dopamine-releasing cells from the midbrains of rats, Sulzer and his coworkers have found that small synaptic vesicles-whose diameters measure in the tens-of-nanometers range and contain as few as 3,000 neurotransmitter molecules-almost always undergo the kiss-and-run type of exocytosis. A larger class of vesicles, which can hold up to 1 million molecules, also can undergo kiss-and-run exocytosis, but most often they deploy the all-or-none tactic.

In their studies of the smaller vesicles, Sulzer's group found that some of the vesicles undergo a single kiss-and-run event, emitting 25-30% of their molecular contents before receding into the cell, and other vesicles undergo a multistep, flickering type of release. In these more complex cases, the small vesicles open and close several times in series, each time releasing more of their neurotransmitter.

"Small synaptic vesicles release about 3,000 to 10,000 molecules over the course of about 100 microseconds," Sulzer says. As the researchers found, each flicker shows up at their carbon-fiber electrodes as an amperometric peak (Nat. Neurosci. 2004, 7, 341). The signal is due to a transfer of electrons from the oxidation of dopamine at the positively biased electrode surface.

This flickering mechanism might enable cells to reuse synaptic vesicles more rapidly than is possible via a more sluggish vesicle-recycling process known to be operative in many types of neurons. This more complicated type of exocytosis also provides a previously unknown mechanism for potentially increasing the number of states the brain can muster, Sulzer says. That's because, compared with an all-or-none release, a flickering mechanism can add more nuance to the chemical signal that one brain cell can send to the next, somewhat like adding inflection to a word when speaking.

Building on that work, Sulzer is investigating how dopamine release is affected by a variety of proteins known to be involved in the making and breaking of synaptic connections, the mainstay of the brain's plasticity and ability to learn in the short term and adapt in the long term. A primary goal of such work is to uncover more details underlying the degradation of dopamine-releasing neurons that occurs in Parkinson's disease, he says.

In one set of studies, the researchers use the electrochemical technique of cyclic voltammetry, which can selectively monitor the presence of oxidizable molecules, to measure dopamine release in brain tissue from a strain of mouse that produces a mutant form of the protein a-synuclein. In humans, this protein mutation is the basis of a heritable form of Parkinson's. In another set of experiments, the researchers are investigating the way the protein VMAT-2, which stands for vesicular monoamine transporter, affects dopamine release in synapses. VMAT-2 is responsible for the uptake of dopamine into synaptic vesicles.

At the Ecole Normale Sup??rieure, in Paris, Christian Amatore and his colleagues have been developing and using electrochemical methods to study exocytosis in cells. In one investigation, they are examining chromaffin cells, which are adrenalin-producing cells located in the adrenal glands above the kidneys. Although these cells are not neurons, researchers sometimes turn to the vesicle-based, adrenalin-release mechanism of these cells as model systems for the kind of exocytosis that neurons exhibit.

Using amperometric methods, investigators can identify and examine "the exact physiochemical nature of all of the individual physiochemical and biological factors which concur to produce vesicular release," Amatore noted last October at a conference on "Charge Transfer at Electrodes and Biological Interfaces," which was convened in Houston by the Welch Foundation.

Most researchers who are now using electrochemical techniques to study the brain rely on cultured cells, tissue slices, and sometimes anesthetized animals, which they can manipulate and stimulate with far more experimental control and clarity than is possible with living, active subjects.

In recent years, however, one of the pioneers of electrochemical brain investigations, R. Mark Wightman of the University of North Carolina, Chapel Hill, has been taking the leap into recording dopamine activity around single cells of living, active rats. Lately, he and his colleagues have been expanding the technique by developing electrodes capable of simultaneously taking electrochemical and electrophysiological measurements-that is, measurements of dopamine release from a cell as well as the cell's firing activity.

In one category of experiments using rats, the researchers place their probes next to individual cells in the nucleus accumbens, a region important in the brain's reward system. Not only does the technique zero in on single cells, but it also can detect the presence of as few as several thousand molecules in the time frame of a single neuronal firing, or about 50 milliseconds, Wightman says. Understanding more of this particular brain region's neurochemical dynamics could lead to insights about addiction to drugs and other stimuli, which is why some of the support for the work comes from the National Institute on Drug Abuse (NIDA).

In fact, a NIDA research program manager, Nancy S. Pilotte, hooked Wightman up in 2001 with another UNC researcher, neurobiologist Regina M. Carelli, for what has turned out to be a happy research marriage. Since the 1980s, when Wightman began developing the application of carbon-fiber electrodes for neurochemical measurements, the questions that had been grabbing him more and more were those that have to do with the relationship between neurotransmitter dynamics and actual animal behavior.

Making such measurements is tricky enough when you're not a card-carrying neuroscientist, but figuring out what the electrochemical data mean is even harder, Wightman says. "The interpretation of the data is the hard part, because no one has had chemical information on this timescale from the brain on this spatial scale before," he says.

That's why Pilotte suggested to Wightman that he contact Carelli. She believed that the two of them working together would have the collective expertise to tell an important neuroscience story that ranges from molecules at synapses of individual brain cells to specific motivation- and addiction-relevant behaviors of animals.

Carelli notes that previous techniques for studying neurochemistry, most notably microdialysis, have been revealing. But the measurements of specific chemicals using these techniques are too slow to discern the neurochemical nuances occurring during a single action potential, a process that unfolds in less than an eye blink.

In a typical microdialysis measurement, a fine hollow probe filled with fluid that mimics the brain's own cerebrospinal fluid is inserted into the brain of an animal, which is then subjected to some stimulus. Chemicals in the vicinity of the probe enter it through a dialysis membrane, and then these chemicals are sorted and identified with chromatography, electrochemical techniques, and other standard laboratory methods. The measurements take minutes, notes Carelli, a time frame that is mismatched with the sorts of motivation- or addiction-related behaviors that she studies. "If you decide to go to the cabinet and get a candy bar, that happens in seconds, not minutes," Carelli points out.

That's where electrochemical methods such as the fast-scan cyclic voltammetry (FSCV) that Wightman has introduced to Carelli, as well as to laboratories as far away as the Netherlands and Japan, have been coming in so well.

"With voltammetry, you can measure things on a few-millisecond timescale, and that was not possible with any other technique," says George V. Rebec of Indiana University, Bloomington. Rebec uses voltammetric techniques to measure ascorbate in the brains of a strain of mouse that models the neurodegenerative malady known as Huntington's disease. He suspects that in certain parts of the brain, there is a relationship between ascorbate, an antioxidant, and the etiology of the disease.

Wightman, Carelli, and their colleagues have used FSCV to measure how dopamine release works in single cells of rats that Carelli had trained to self-administer cocaine. The drug inhibits the uptake of dopamine by neurons. When that happens, there's more dopamine around for a voltammetry electrode to oxidize.

For Carelli, protocols that use isolated cells, tissue slices, or even animals in anesthesia-induced sleep can't be as valuable as experiments with animals on the go for answering questions about addiction, seeking mates, and other motivated behaviors. Carelli says, "If I want to understand the biological basis of motivated behavior and how drugs of abuse lead to addiction, then I want to measure chemical events in living, awake animals engaged in those behaviors."

For Wightman, these studies begin the way hundreds of others have begun for him over the past 20 years. "We take one 10-µm carbon fiber and put it into a capillary tube so that a little carbon is sticking out of the end," he says. Under a microscope, the arrangement looks like a mechanical pen with a graphite "lead" sticking out of the end. When a particular range of electrical potentials is applied to the fiber, it will selectively oxidize molecules that give up an electron in that potential range. This is why the same basic setup can be used to measure dopamine, serotonin, adrenalin, histamine, and a portfolio of other molecules.

This also is why researchers must be aware of which other oxidizable molecules, including ascorbate, might be near the electrode and why the technique is not universally applicable for measuring any neurochemical that a researcher might be interested in.

For these live-animal studies, Carelli first trains a group of test rats in an environment that enables them to self-administer cocaine by using a lever that triggers an intravenous injection. The experiments also include a control group of "naive" rats that have not been trained this way and so are not addicted to cocaine.

The next task with each animal is to surgically implant one of Wightman's carbon-fiber electrodes into a region of the animal's brain, such as the nucleus accumbens, that contains dopamine-releasing neurons. At the same time in some experiments, another stimulating electrode is implanted into a different brain region, the ventral tegmental area, whose neurons make connections with those in the nucleus accumbens. When stimulated, this brain area triggers dopamine release from cells in the nucleus accumbens.

Several days after the electrode-implantation surgery, the animals are ready for voltammetry experiments. With the help of these animals, the researchers have conducted a large and growing portfolio of experiments. In some, rats that had been trained to self-deliver cocaine before the electrode-implant surgery were allowed to do so on the test day. During the test sessions, which could last for a few hours, voltammetric recordings were taken at a rate of about 10 per second, resulting in thousands upon thousands of data sets.

In other experiments, rats that had been previously exposed to cocaine were given the drug on a noncontingent basis during the test sessions-that is, cocaine administration was under the control of the experimenter and not dependent upon an animal behavior like lever pressing. Some animals tested had never received cocaine before the voltammetry runs. In some experiments, the researchers measured dopamine release following gentle jolts with a stimulating electrode rather than with the administration of cocaine.

During the measurement of each voltammogram, the voltage of the probe was rapidly cycled up and back over the course of about 100 milliseconds from about -0.4 V to 1.3 V. That's a voltage range in which dopamine oxidizes, and so, if the neurochemical is near or on the electrode, it will release electrons to create a current.

When sequences of hundreds of cyclic voltammograms are aligned and plotted onto panels, and when current changes are color coded, the result is a colorful map that reflects both large and small dopamine concentration changes before and after lever presses, electrical stimulations, and noncontingent administrations of cocaine and changes in response to other experimental stimuli and conditions.

These maps are reminiscent of abstract art paintings, but they provide a quickly graspable yet nuanced and starkly visible sense of at least some of the neurochemistry that is going on in what Carelli believes to be important facets of the development and maintenance of addiction.

These maps even appear to indicate what the researchers interpret as drug craving, she says, because some of the "dopamine transients" (see cover) that the researchers have seen occur just prior to the pressing of a cocaine-releasing lever.

Because cocaine inhibits the reuptake of dopamine released by cells, the dopamine signal increases noticeably after a rat receives either self-administered or researcher-administered cocaine. These spikes, which peak about 90 seconds after cocaine delivery, have been observed to some degree in previous experiments. However, the increased time resolution of FSCV compared with other techniques has enabled Wightman, Carelli, and their colleagues to discern finer structure in the dopamine dynamics than had been possible before.

One key finding is that animals receiving cocaine at the will of the researcher, and not at times that they could control with a lever, displayed many more transient dopamine release events that had no obvious correlation with any environmental stimuli. It was as if each animal's brain was anticipating a cocaine treat at any moment and was saying, "Maybe now, maybe now," each time releasing a little dopamine.

"We hope we are learning things that could be practical," Carelli says, noting that details like those in the dopamine signaling that her collaboration with Wightman is chronicling could provide clues to new types of interventions that might be useful to fight addiction.

To take these and related measurements even further, the researchers have been developing and using electrodes with which they can quickly toggle between electrochemical measurements of dopamine and electrophysiological measurements of the neuron releasing the dopamine. "This enables us to get a feel for the effect dopamine is having on the cell's electrical activity," Carelli says. They also now are developing multibarrel electrodes that will enable them to deliver specific neuroactive agents to the very cells at which they are measuring dopamine.

At Illinois State University, Normal, John E. Baur and his colleagues are adapting a technique called scanning electrochemical microscopy (SECM) to simultaneously map out the topography of individual cells while measuring concentrations of specific chemicals at different locations just outside the cell.

"We want to be able to do fast-cycle voltammetry at each pixel," Baur says, referring to each of many tiny spots located just above a cell's surface. One goal, Baur says, is to have a tool that will enable scientists to monitor the growth and development of model neurons while also tracking the types and concentrations of chemicals that they release over time.

A big challenge here, Baur says, is to keep the tip of the scanning electrochemical microscope at a constant distance from the irregular surface of the cell as the tip scans over the cell. Without that constant separation, he notes, the relationship between the voltammetric signal and the concentration of neurochemicals the probe is detecting cannot be delineated with confidence.

To be sure, applying electrochemical tools and techniques to tease out more of the fine details of what brain cells are doing is hard and fraught with pitfalls. Rebec, who worked with Wightman at Indiana University in the 1980s, recalls how difficult it was in the past to distinguish an electrical signal due to dopamine oxidation from one due to oxidation of other molecules such as ascorbate. "Electrochemistry took a back seat in neuroscience until we knew what we were looking at," Rebec remarks.

In some ways, the level of detail delivered by electrochemical studies of single brain cells, even of single synaptic vesicles of single brain cells, is daunting, researchers maintain. "You can be too 'single-cell-aholic,' " quips Andrew G. Ewing of Pennsylvania State University. He has been using electrophoretic separation techniques coupled with electrochemical detection to analyze individual homogenized fruit fly heads for their content of biogenic amines, such as dopamine and histidine.

Using SECM, FSCV, and other electrochemical techniques for monitoring neurochemistry at the level of single cells has yet to become commonplace in neuroscience labs. It takes an unusual spectrum of interdisciplinary skills and know-how to make the tools and measurements as well as to make much behavioral and psychological sense of the raw electrochemical data, which so often look like unassuming closed loops with odd shapes or blips of an electrocardiogram.

Even so, Wightman says, there's a world of discovery awaiting those willing to give it an electrochemical go. To the tip of an electrode situated a few micrometers away from the end of a single neuron, the brain is a vast territory with a chemical diversity that has yet to be fully documented. It's a mind-numbing prospect, Wightman concedes, but he says there's an irresistible upside to it: "Where you stick an electrode, you will make a discovery."