"Analytical chemistry in the brain is very challenging," symposium co-organizer R. Mark Wightman, the W. R. Kenan Jr. Professor of Chemistry at the University of North Carolina, Chapel Hill, told C&EN. "There are a million compounds, and you want to pick out and look at one or two. Once you're able to identify them, you want to understand what they're doing."

Christian Amatore, professor of chemistry at Ecole Normale Superieure, Paris, described electrochemical monitoring of exocytosis, the process of releasing materials such as neurotransmitters from cells, using carbon fiber microelectrodes.

Five independent physicochemical stages govern exocytosis, Amatore said. In his presentation, he focused on the stages in which the vesicle docks to the cell membrane, forming a fusion pore, and in which the vesicle and cell membranes fully fuse. These stages affect the shape of the spikes observed during electrochemical monitoring of neurotransmitters. When the fusion pore forms, a small amount of the vesicle contents is released, with a large spike of released material following full fusion. Amatore pointed out that in electrochemical monitoring there is not always a sharp transition between the fusion pore and full fusion.

Wightman uses the electrochemical technique cyclic voltammetry to monitor the release of dopamine, which is thought to be part of the brain's reward system, on a subsecond timescale. He also uses microelectrodes fabricated from carbon fibers. Wightman was a pioneer in the development of the carbon microelectrodes that many chemists use to monitor neurochemistry.

One of the challenges is making sure that he's really looking at dopamine. The cyclic voltammetric signature of dopamine is similar to that of another neurotransmitter, norepinephrine. However, Wightman can distinguish dopamine by measuring in the region of the brain known as the nucleus accumbens, where there is no interference from norepinephrine.

Dopamine neurons can undergo "tonic firing" or "phasic firing," Wightman said. Tonic firing occurs at a frequency of about 5 to 10 Hz, and it represents the basal firing rate. In contrast, phasic firing occurs in short, fast bursts that last less than half a second.

"Before the development of these carbon fiber electrodes, there was never a way to follow these changes on subsecond timescales," Wightman told C&EN. "People didn't know what dopamine was doing on a fast timescale."

BY TRAINING RATS to respond to audiovisual cues that are associated with a reward, in this case the self-administration of cocaine, Wightman's research is showing that dopamine can be elevated in anticipation of receiving a reward.

When a rat is placed in a chamber with a lever that dispenses cocaine, the rat first presses the lever frequently to load up with cocaine and thereafter presses the lever at regular intervals. After the rat presses the lever, the amount of dopamine increases.

However, an interesting thing happens when the rats learn to associate the cues with the administration of the cocaine. They experience an increase in dopamine when they approach the lever but before they press it. This dopamine increase during "approach behavior" doesn't happen with untrained animals, so it's not caused by just the cues.

"Before, people thought that dopamine being elevated was a reward in itself," he said. "We're looking at these real fast things, which deal with the anticipation or the alerting of the animal that something is going to happen."

Jonathan V. Sweedler, professor of chemistry at the University of Illinois, Urbana-Champaign, is trying to identify previously unknown catabolites of neurotransmitters. "You want a fairly information-rich analytical technique," Sweedler told C&EN. "We're using a very selective detector, using a UV excitation wavelength that only looks for certain types of compounds and is effective for things like indolamines and catecholamines, which happen to be neurotransmitters of interest."

His group uses capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) detection to study the serotonin content in identified neurons in the marine snails Aplysia californica and Pleurobranchaea californica. Sweedler likes to use these simple marine invertebrates because they have a relatively small number--approximately 10,000--of neurons, which are physiologically well defined.

In one example, Sweedler and his collaborators used CE-LIF to study the difference between feeding and quiescence in Pleurobranchaea. Serotonin levels track the feeding threshold, Sweedler said. The snails were paired according to size and starved for about a month. Then one of the slugs in each pair was fed. The hungry animals had a fourfold higher serotonin level in one particular neuron, the metacerebral cell.

Sweedler and his coworkers have also been able to identify new catabolites in the sea snails. They found that sometimes nitric oxide causes the formation of serotonin dimers. So far, Sweedler doesn't know how often this dimer forms or what its function is. Sweedler also identified serotonin sulfate catabolites with unknown function. He speculates they may play a role in regulating global serotonin levels in the brain.

Anne M. Andrews, chemistry professor at Pennsylvania State University and symposium co-organizer, also studies serotonin, but she studies it in genetically engineered knockout mice that don't express the gene for the serotonin transporter. Her ultimate interest is in how serotonin is involved in psychiatric and degenerative disorders. Serotonin has been implicated in a number of psychiatric disorders, such as depression and anxiety, and in diseases associated with aging, such as Parkinson's and Alzheimer's.

	Sweedler	Amatore
	PHOTOS BY CELIA HENRY
	Andrews	Shippy

ANDREWS USES a variety of techniques depending on the information she's looking for. Microdialysis allows her to measure basal levels of synaptic serotonin in the brain. However, microdialysis is slow, so she can't use it to distinguish between release and reuptake of serotonin as separate processes. For faster measurements, she also uses carbon fiber microelectrodes.

She uses the carbon electrodes in an ex vivo preparation known as synaptosomes, which are liposomes prepared from the animal's brain tissue. "For 40 years, biochemists and neuroscientists have been using this preparation to study uptake and receptor binding," Andrews told C&EN. Traditionally, synaptosomes are used with radioactively tagged neurotransmitters.

However, when Andrews and her colleagues weren't able to see any differences in the rate of serotonin uptake between wild-type mice and mice that had one copy of their serotonin transporter gene inactivated (known as heterozygotes), they concluded that the traditional method was not sensitive enough. "We'd already shown that the protein expression was decreased by 50%. In addition, those animals had a behavioral phenotype," she said. "We began to suspect that the classic radiometric assay was not sufficiently sensitive to detect modest changes."

When they started using electrochemical detection methods, Andrews thought the measurements should be done in synaptosomes so that they could be compared with the traditional radiometric method. Another reason for choosing synaptosomes is that, unlike dopamine, serotonin is not restricted to certain parts of the brain. "When we use chronoamperometry in synaptosomes, the signal that we're generating comes from serotonin that we've injected," Andrews told C&EN. "We know what the source of our electrochemical signal is under those conditions."

With chronoamperometry, Andrews is getting 60 measurements per minute. At first, the serotonin uptake rates seemed slow, but then Andrews realized that they should bubble oxygen through the synaptosome solution, which caused a 100-fold increase in uptake.

"We think the fact that we couldn't detect changes in heterozygote animals was a combination of the fact that the radiometric technique was insensitive and we weren't doing the experiment under biologically relevant conditions," Andrews told C&EN.

SCOTT A. SHIPPY, an assistant chemistry professor at the University of Illinois, Chicago, is trying to breathe new life into a technique known as push-pull perfusion, which had fallen out of favor because the large probes required high flow rates and poked holes in the brain. Push-pull probes consist of concentric cylinders. An infusion solution enters through a longer inner cylinder, and the sample is recovered through a shorter outer cylinder.

Shippy needed to miniaturize the probes and reduce the flow rate used in push-pull perfusion. He is currently using flow rates of 10 to 15 nL per minute. He also switched the cylinders so that infusion is through the outer cylinder and recovery is through the inner cylinder. In his setup, the inner and outer cylinders end at the same place, which may provide better spatial resolution, he said.

Shippy used the new probes to measure the neurotransmitter glutamate in rats. The basal glutamate values that he measured matched those in the literature, he said. In addition, the probe caused no detectable tissue damage.

Although the speakers at the symposium have made progress toward understanding brain chemistry, there's still a long way to go. "There is a great need for increased development of analytical methods for measuring brain chemistry in temporally resolved ways," Andrews said.

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