Report
PROBING BRAIN CHEMISTRY
U
nderstanding the chemistry of the brain has been a longtime goal. However, because of the small size of synapses and the inaccessibility of the brain, the task of making meaningful chemical measurements has not always been easy. Direct assessment of tissue content for neurotransmitters metabolites and other species has been done for many years and much has been learned from
Voltammetry Comes of Age
However, there are several drawbacks to tissue assays. Aside from the fact that only one time point is obtained from a subject, tissue content does not yield information about compartmentation of the neurotransmitter among the various structures of the brain such as neurons, glia, cytoplasm, vesicles, and extracellular fluid. Because communication is achieved via chemicals released by one cell to stimulate receptors on the surface of another cell it is primarily the compartment outside the cells where observations desired particularly the release and removal of transmitters through a treelike network called denTwenty-one years ago, Ralph Adams in- drites and transmit it in the form of electritroduced in vivo voltammetry (2)) and the cal impulses, or action potentials, along a field has evolved to the point that it is appro- cablelike axon to other nerves. At the end of the axon, the message is relayed to priate to say that, at 21, it has come of age. In this Report, we describe the research other cells at points of contact called synapses, where the electrical message highlights since Adams' article and briefly survey the use of voltammetric methods to causes the release of a chemical neurotransmitter that diffuses across a small gap probe brain chemistry. More complete reviews and background can be found elsewhere (2-4). The mammalian brain contains ~ 1011 J o n a t h a n A. S t a m f o r d nerve cells or neurons, which assimilate London Hospital Medical College and process information. These neurons J o s e p h B. J u s t i c e , Jr. receive information from other cells Emory University
Voltammetry has been solving the mysteriesofthe brain and its functions for 21 years
0003 - 2700/96/0368 -359A/$12.00/0 © 1996 American Chemical Society
to another neuron. The neurotransmitter binds to and activates a receptor on this second neuron and contributes to the formation of another action potential, repeating the cycle. Each neuron may have about 1000 such synapses and thus can integrate information received from many other cells. Figure 1 is a diagram of a nerve terminal. Neurotransmission, the conversion of an electrical impulse to a chemical event and then to another electrical event, is extremely rapid. Action potentials and neurotransmitter release typically last about a millisecond. In many ways, the release and actions of neurotransmitters represent the bricks with which the internal representation of the external world is built Early studies of the chemistry of release and uptake included using metabolites as an indirect index of neuronal activity. A more direct observation of released transmitters was achieved using push-pull perfusion in which concentric metal tubes were implanted in the brain and used to sample the extracellular fluid. New information was obtained, but the method caused severe tissue damage, often needed radioactive materials to achieve the necessary sensitivity and was slow relative to the speed of neurotransmission itself In many respects the field was ripe for a breakthrough In vivo voltammetry
In the late 60s and early 70s, Adams had been studying the solution electrochemistry of an important class of neurotransmitters, the catecholamines. He reasoned
Analytical Chemistry News & Features, June 1, 1996 3 5 9 A
Report that a miniaturized carbon paste voltammetric electrode could be implanted in the brain and oxidation currents from catecholamines could be observed. The first experiments produced oxidation currents, but several other oxidizable molecules, notably ascorbic acid, obscured the currents from catecholamines. Another problem was that the extracellular concentration of the deaminated transmitter metabolites was often 1000-fold higher than the parent transmitter. For example, signals from dihydroxyphenylacetic acid often swamped the current from dopamine. At the time that Adams was doing hii pioneering work in the United States, Francois Gonon and his colleagues in France had been investigating electrodes made of other carbon-based materials for detecting catecholamines (5). By encasing a carbon fiber in a glass micropipette, they created an electrode that was small enough to be implanted into brain tissue and, in combinatton with differential pulse voltammetry (DFV), could detect catechol- and indole-based compounds separately. Although the work of Adams and others showed that voltammetric measurements in vivo were possible, selectivity and sensitivity needed to be improved. Many approaches were tried, including numerous waveforms, from simple chronoamperometry through DPV and other more exotic extensions. Two breakthroughs in selectivity were achieved with the use of Nation coating (6), an anionic polymer that reduced interference from ascorbic acid and acidic metabolites and with electrochemical pretreatment (7) which shifted the oxidation potentials of interferences Both annroaches are now widely used
Transmitter release and uptake Rather than stick to the original goal of trying to detect basal dopamine release, Wightman chose (perhaps out of frustration) to stimulate the tissue electrically and measure the release and subsequent removal of neurotransmitter in the extracellular space. This simple decision not only liberated electrochemists from a difficult task but also allowed voltammetric techniques to be applied to studying the actual dynamics of neurotransmission. Basal levels of catecholamines have since been routinely measured with microdialy360 A
Figure 1 . Simplified diagram of a catecholamine nerve terminal. The neurotransmitter is synthesized and stored in vesicles that fuse with the membrane to release the transmitter into the synaptic space. Once released, it binds to postsynaptic receptors and autoreceptors. Uptake and diffusion remove the transmitter to terminate the stimulation of the postsynaptic cell. Transmitter that is taken up may be recycled into storage vesicles or metabolized.
sis by using an electrochemical detector for HPLC (8), another development from Adams' lab. In 1984, Wightman and Stamford used fast cyclic voltammetry (FCV) to detect dopamine released during electrical stimulation and subsequently removed by uptake (9). The ability of these fast techniques to make many recordings per second has allowed real-time measurement of neurotransmitter re-uptake to be examined. These measurements have shown just how fast these processes really are (10,11) and thus provide genuine insights into their roles in neurotransmission. Interestingly, uptake of a given transmitter often varies in different brain regions (12,13), and thus its role may differ. For instance, less uptake or fewer uptake sites infers that released transmitter may diffuse further before being removed from the biophase. This tendency, in turn, means that the transmitter may have actions at sites more distant from the synapse. Initially, transmitter release was recorded after relatively large stimulations (tens or hundreds of pulses lasting many seconds) released most of the available pool of neurotransmitter (14-16). For example, 10 s of stimulation increases the extracellular dopamine from low-nanomolar to micromolar concentrations a
Analytical Chemistry News & Features, June 1, 1996
1000-fold increase that is not representative of physiological conditions. When the stimulation is terminated, uptake removes the dopamine in a few seconds. Subtler stimulations are now used to probe the intricacies of neurotransmitter release. Shorter stimulus trains (only a few pulses) lasting less than a second released less of the available transmitter and thus could be repeated more often and could provide temporal information about the onset and offset of drug action (11). The ultimate extension of this is a stimulus train of only a single pulse (17)) Under normal physiological conditions, an action potential causes the release of neurotransmitter, which, in addition to its action on other cells, acts on autoreceptors on the nerve terminal to reduce the release of transmitter by subsequent action potentials (Figure 2). This autoinhibition phenomenon is a fundamental means by which neuronal communication is self-limited. On the other hand if the autoreceptors are blocked by an antagonist autoinhibition is suppressed and transmitter release is thus elevated causing excessive stimulation of postsynaptic receptors However, a single pulse should not be subject to autoinhibition because there is no preceding pulse to release transmitter. With the demonstration that dopamine release could be detected voltammetrically following a single pulse of stimulation (17), the hypothesis was directly testable. Limberger and colleagues (18) showed that dopamine released on a single pulse inhibited dopamine release on another pulse delivered a second later. This effect could be prevented if the autoreceptors were blocked by a dopamine antagonist Electrodes and the brain While learning about the processes of neurotransmission, we have also learned about the way in which the brain responds to implanted electrodes. Unlike an electrochemical cell, the brain is an electrically and chemically active environment. The implantation of an electrode is traumatic to brain cells, and it is intuitively obvious that the smaller the electrode the less damage that occurs. Modern carbon fiber microelectrodes, only —10 um in diameter, cause much less damage than the wider ~250-um carbon paste electrodes
used 20 years ago. The fact that carbon fibers can also be used to monitor the electrical activity of individual nerve cells for prolonged periods is further evidence of their safe use. The smaller current flow through microelectrodes is much safer and, when combined with scanning techniques, provides as much information as large electrodes with DPV and other waveforms. Smaller electrodes also minimize artifacts (19). Interestingly, the size of the electrode can alter the species detected. For example, larger electrodes may disrupt the bloodbrain barrier and allow blood-borne compounds to be detected or they can evoke a local gliosis (tissue reaction to a foreign object) (20). The consensus is that small electrodes and nonpulsatile recording methods form the best strategy to minimize interference with brain function. We now also know how the neuronal microenvironment may influence electrodes and voltammetric measurements (21). Although in many ways an excellent supporr electrolyte, the extracellular fluid (ECF) does contain protein that may adsorb to electrode surfaces, impede electron transfer, and thus decrease electrode sensitivity. This occurs more or less immediately following electrode implantation with little additional deterioration as time passes (22), but it can bb partially circumvented by covering the electrode surface with permselective coatings such as Nation In addition to enhancing selectivity for the monoamines over their anionic metabolites these coatings also orobablv reduce ffoling by protein Poisoning of electrodes by oxidation products is more difficult to negate. Phenolic and hydroxyindole oxidation products, such as those arising from serotonin, are particularly troublesome because they tend to be insoluble and may form afilmat the electrode surface. Although there is little control over the concentration of these compounds detected in vivo, we now know that electrodes should not be exposed to high concentrations when calibrating. The time of an electrode IYI3V
also be affected if it is covered in brain tissue or by a film that can act as a physical barrier through which amines must diffuse thereby causing artificially slow response times to changes in concentration It was recognized early that a pool of ECF forms at the tip of an implanted elec-
trode (23), and that standard solution diffusion coefficients are applicable within this zone. However, transmitter diffusing through the ECF into the pool must pass between nerve cells. This tortuous route means that the functional diffusion coefficient is decreased. For long measurements that deplete the electrode tip pool of transmitter by electrolysis, the pool can be replenished only by diffusion on this slower path thereby making fast measurements advantageous.
Figure 2. Using brain slices with voltammetry. (a) A stimulating electrode and a working electrode are implanted in a brain slice. Following stimulation, transmitter release occurs in a small region of the brain slice (circle) and is detected by the working electrode, (b) Dopamine (DA) release on two stimulation pulses applied to the slice, 2 s apart. Dopamine release on the second pulse is lower than on the first, (c) Schematic representation of nerve terminals with dopamine (dots). On stimulation 1, dopamine is not yet released. However, on stimulation 2, dopamine released by the previous pulse has activated the autoreceptor (rectangle) which, in turn, reduces the amount of dopamine released by the second pulse.
The desire to minimize tissue damage has meant that, for many of today's applications, the carbonfibermicroelectrode is the working electrode of choice. The carbon fiber extending beyond the insulation may be cut or beveled such that the active surface is a disc, cylinder, or ellipse. These electrodes may then be used as prepared, may be electrically pretreated, or the surface may be coated with Nation (6). Real-time voltammetry
FCV came about because smaller carbon fiber electrodes allowed faster electrochemical measurements to be taken. The voltage scan rate was increased and, rather than lasting a couple of minutes, each scan took only 20 ms and thus could be repeated manytimesper second. The features that distinguish FCV from classic voltammetric methods are the speed of the measurement and the size of the electrode used. Whereas most "beaker-type" electrochemical measurements using electrodes up to 3 mm in diameter last several seconds or minutes (10 mV/s is a typical scan rate), an FCV scan takes only 20 ms (~ 400 V/s) and is made at a carbonfibermicroelectrode ~ 8 um in diameter. These measurements can be repeated many times per second and thus give near-real-time detection of transmitter release In FCV, background current signals prior to a stimulation or "event" are digitally subtracted from those obtained during a transmitter release event. Thus, current common to both is eliminated, and only the faradaic current from the oxidized species remains. Despite the high scan rate of the applied voltage waveform, FCV does not affect neuronal activity (20). Indeed, FCV may be combined with electrophysiological recording at the same electrode (24 25). In other words it is possible to probe the electrical and neurochemical activity of the cells simultaneously This capability has exeatly enhanced the quality of data acquired by each annroach used in isolation and has allowpH ttie relationship hptwppn prp and
to be investigated FCV is used mainly to monitor the release and uptake of endogenous monoamine neurotransmitters in vivo (10,11) and in vitro (17,26,27). One advantage of FCV has been to shift the emphasis away
Analytical Chemistry News & Features, June 1, 1996 361 A
Report from metabolites (as indices of transmitter release) and to focus attention on the neurotransmitters themselves.
the penetration of different types of dopamine nerve terminals in the medial part of the striatum (30).
Beyond the whole animal
Voltammetry w i t h cells
Parallel to many of the technological developments has been a dramatic extension of the range of voltammetric applications. For instance, the advent of brain slices (28) has revolutionized functional neurochemistry. The brain slice allows the user to control most features of the physiological environment (temperature, pH, and ionic composition) in a way that is not possible in vivo. True concentrationresponse curves can be constructed without the distraction of actions mediated peripherally or at other locations within the central nervous system Studies in brain slices are also conducted without anaesthetic agents to avoid unwanted drug interactions The use of brain slices also allows the actions (inhibition or stimulation
Although slices are themselves a simplified system in relation to the whole animal, it is possible to take an even more reductionist stance. The cell is a fundamental unit and level of analysis in biological systems, and the ability to analyze the chemical constituents of single cells is of considerable interest. The small sample size is the limiting factor, both with respect to detection limits and sample handling. The development of a "chemical neurophysiology" moved much closer to realization with the recording of transmitter release from single cells (3D
rvf relpnse or uptakp^ of many rimers that
cannot be studied in • L I
animals cause they are either too toxic or they do t r a h th brai ) to be e aminerl A common feature of many previous nonvoltammetric studies in slices is the use of radiolabeling to tag the tissue neurotransmitter pool. There are serious limitations to this approach. This technique is based on the erroneous assumption that exogenous radiolabel is released by the tissue in an exactly synchronous fashion with the endogenous transmitter. In addition, it is often necessary to block neurotransmitter re-uptake to obtain detectable transmitter efflux in these experiments. Lastly these kinds of studies have negligible SDatial resolution it is sary to evoke transmitter release from most of the tissue to obtain detectable lPVPIS
An ideal method would detect spatially resolved, calcium-dependent release of the endogenous transmitter, without the artificial circumstance of uptake blockade. In many respects, voltammetry measures up well to these ideals and a wealth of information has been obtained about the dynamics of transmitter release and reuptake. Because the positioning of electrodes in brain slices is done visually, electrodes can be placed accurately in very small nuclei that are difficult to access stereotaxically in the whole animal. This 362 A
Figure 3. Using voltammetry to measure exocytosis of an adrenal chromaffin cell. (a) A beveled carbon fiber microelectrode is shown immediately above the cell. On stimulation, the vesicles fuse with the cell membrane, and packets of catecholamine release and diffuse to the recording electrode. (b) The current output is held at a fixed potential (+0.65V). The "spikes" correspond to the fusion of individual vesicles, (c) Background current-corrected fast cyclic voltammograms (right) of the compound detected at the beginning and peak of an individual spike. In each case, the signal consists of a single redox couple, characteristic of a catecholamine. (Courtesy of Mark Wightman.)
gives the method excellent spatial and temporal resolution. Like in vivo voltammetry, the first recorded voltammetric experiments in brain slices to look at K+-evoked dopamine efflux in the striatum (brain region rich in dopamine nerve terminals) were conducted in Adams' laboratory, mainly to shed light on data obtained in vivo. Since then, the brain slice-voltammetry methodologies have been used to measure ascorbic acid (29), serotonin (26)) and norepinephrine (27) in a much larger range of nuclei. In addition to pharmacological studies, carbon fiber microelectrodes can be used to conduct spatial studies to make a functional map of brain areas. For example, this approach has been used to map
Analytical Chemistry News & Features, June 1, 1996
This endeavor has now reached the stage that individual exocytotic release events, the "quanta" of neurotransmissions, can be measured. Figure 3 is such an example for an adrenal chromaffin cell that is, in essence, a nerve terminal. These cells possess most of the transmitter release machinery of a neuron and, when stimulated, release catecholamine (mainly norepinephrine and epinephrine). Stimulation of the cell with K+ or nicotine results in a series of current transients at the electrode. These transients arise from individual vesicles releasing their contents of neurotransmitter into the extracellular space. Wightman and coworkers have examined the temporal characteristics of these observations with respect to diffusional broadening as a function of electrode distance from the cell surface (32,33). Important characteristics of vesicular release can be studied including delay in vesicle fusion (34) and the quantal nature of the release (35 36) Of course you don't have to park the electrode outside the cell. Because carbon fiber microelectrodes with tips smaller than 1 um are available, it is possible to penetrate the cell membrane and study events within the cell (37). Surprisingly, cells tolerate this intrusion rather well and appear to seal around the electrode tip. Just as studies on transmitter release have benefited from a reductionist approach, so have studies on uptake. A new application of an old method, rotating disk electrode (RDE) voltammetry, is turning out to be a useful way to examine cate-
cholamine uptake. Schenk has shown that RDE voltammetry can be used to follow the disappearance of catecholamines from a rapidly stirred suspension of synaptosomes or tissue homogenate (38,39). The significant feature of the method is that a complete time course of uptake is obtained, including the first few seconds of uptake after the addition of substrate. In addition effects of diffusion are minimized by the rapid mixing. It is possible to obtain an excellent measure of the initial rate as well as data on processes that develop later as substrate accumulates inside the nerve terminals The data can be analyzed in detail for information on the kinetics of the transport process Also becau^p mncf «nh
