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Electrophysiology Microdialysis Electrochemical Techniques
B. Jill Venton
Measuring chemical events in neurotransmission is challenging because fast measurements of small amounts of neurotransmitters and neuromodulators are needed to track the dynamics of chemical changes accurately. In this article, we outline the basics of three popular methods for measuring neurochemical changes: electrophysiology, microdialysis, and electrochemistry. Electrophysiological techniques measure changes in membrane potentials associated with neurotransmission. These methods are often used to measure receptor-gated ion channel currents and are popular for neuropharmacology studies. Microdialysis is a sampling technique that can be used to monitor basal levels of neurotransmitters directly. When coupled to a separation technique, microdialysis is advantageous because it can be used to detect virtually any compound in the brain. Electrochemical techniques are popular because microelectrodes allow rapid, direct detection of neurotransmitters with minimal tissue disturbance. Although the analytes must be electroactive, electrochemistry has been used successfully to monitor neurochemical changes in various preparations, from single cells to behaving animals. Future research in monitoring neurochemical events will include improving the temporal resolution, spatial resolution, and selectivity of measurements.
Neurotransmission is the transfer of an informational signal, a chemical messenger, between two neurons. The traditional picture of synaptic transmission is shown in Fig. 1. A terminal from one neuron forms a synapse with a dendrite of another neuron. After the neurotransmitter is synthesized in the perikaryon of the terminal, it is packaged into specialized synaptic vesicles. The vesicles range in size from about 50 nm to 100 nm and can store between 3000 and 30,000 neurotransmitter molecules (1). An action potential propagating to the terminal changes the cell membrane potential and can induce the fusion of vesicles to the synaptic membrane. This fusion results in exocytosis, the coordinated release of transmitter molecules into the synapse (Fig. 1). Transmitters can then bind to postsynaptic receptors and activate signaling pathways through GTP-binding proteins or open gated ion channels, which leads to localized changes in membrane potential (2). Membrane potentials, ionic currents, and action potentials can be measured using electrophysiology techniques.
Released neurotransmitters can have fates other than binding postsynaptic receptors. Transmitters can bind to presynaptic receptors that act as a feedback loop to regulate additional release of the chemical messengers. Transporters take up neurotransmitters back into the neuron and clear them from the extracellular space. Neurotransmission can also be ended by enzymatic degradation of transmitters, although this process is kinetically slower than uptake. Although the traditional picture of neurotransmission is of short-range signaling, neurotransmitters can also diffuse out of the synapse and act at distal targets, which allows longer distance signaling (3). This process is called volume transmission, and extracellular detection methods such as microdialysis and microelectrodes detect these extrasynaptic concentrations. 1
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
Presynaptic Receptors
Vesicles
Neurotransmitters
Postsynaptic Receptors
Figure 1 General concept representation of synaptic transmission. Vesicles that contain neurotransmitter molecules dock to the cell membranes and release their contents into the synapse by exocytosis. Neurotransmitters can diffuse across the synapse and bind to postsynaptic receptors or can diffuse out of the synapse. This extrasynaptic neurotransmitter can bind to presynaptic receptors, diffuse away and activate receptors on distal neurons, or be cleared from the extracellular space by transporters.
Electrophysiology
Electrophysiological techniques measure changes in potentials associated with neurotransmission. The potential of the neuronal membrane is controlled by ionic concentrations, which are regulated by the active transport of ions. As postsynaptic dendrites receive neurotransmitter signals, receptor-gated Na+ and Ca+ channels are activated, which allows inux down the ionic gradient. Membrane depolarization occurs as cations enter the cell. Once the depolarization reaches a certain threshold, successive Na+ and Ca2+ ion channels open along the axon in a domino fashion to propagate the electrical signal. This propagation of voltage is called an action potential, and it will not occur unless a threshold depolarization is reached. After maximal depolarization, Na+ and Ca2+ ion channels begin to close, and simultaneously, K+ ion channels open and K+ efuxes out of the nerve cell. This process reverses the polarization, and because of the length of time the K+ channels stay open, the abundance of cations outside the cell causes a hyperpolarization of the membrane potential. Transmembrane movement of ions is controlled by gated ion channels and can be represented by a simple RC circuit, in which the ion channels act as resistors and the cell membrane acts as a capacitor. Hodgkin and Huxley, who won the 1963 Nobel Prize for Physiology or Medicine for their work, quantied these transmembrane voltage changes in the axon of a giant squid and determined the time-dependent behavior of Na + and K+ channels. Current electrophysiological studies measure neurogenic changes through various intracellular and extracellular techniques. Although these techniques are diverse, common intracellular techniques include the two-electrode voltage clamp, patch clamp, and whole cell methods that measure ion channel currents and common extracellular techniques that measure voltage changes outside cells. 2
Patch clamp
Some of the most popular electrophysiology methods currently used are patch clamp techniques, in which individual ion channels can be studied. Unlike the two-electrode methods described above, these methods use a single electrode to measure voltage changes. A glass pipette electrode with a at, open tip is placed onto a cell, and piece of membrane that contains an ion channel is sucked into the pipette tip. This action allows the direct current of ions moving through channels to be measured. Although the pipette tip has a high resistance, negative pressure
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
(a) 50 V (mV) 30 20 10 50 mV 180 nA 5 ms (b) 200 50 100 150 I (nA) 8/11 cells 25 ms 10 20 30 40 50
EPSC
2pA
Figure 2 a) Two-electrode voltage clamp experiment. The cell potential was held at 35 mV, depolarizing steps from 35 to +20 mV were made (top left), and resultant inward currents from Ca2+ channels were measured for each step (bottom left). The right graph shows an I-V curve for nematode muscle Ca2+ ion channels. (Data reprinted from Ref. (4) by permission of Macmillan Publishers Ltd.) b) Whole cell experiment. Representative trace of an individual EPSC before (left) and after (right) application of an adenosine A2A receptor agonist CGS 21680. The agonist causes the ESPC amplitude to decrease. (Data reprinted from Ref. (5) by permission of Elsevier.) c) Inside-out experiment. A continuous single-channel current is shown from in an inside-out membrane patch of a rat cultured hippocampal neuron before and after addition of diazepam. The top trace shows currents induced by 5M GABA. The bottom four traces show currents induced after a 1M solution of diazepam is introduced. A gradual increase in single-channel current amplitude occurs. (Data reprinted from Ref. (8) by permission of Macmillan Publishers Ltd.)
is applied to form a seal with a gigaohm resistance, which is commonly referred to as a gigaseal. The pipette tip is usually lled with a solution that approximates intracellular uid. Two types of experiments can be performed once the pipette electrode is in place: voltage clamp or current clamp. Similar to two-electrode voltage clamp methods described above, in voltage clamp mode the membrane potential is held at a constant voltage, and current through the ion channels is measured. In current clamp mode, the current is kept constant, and voltage changes are measured. To identify which types of channels contribute to the signal, electrophysiology techniques are commonly used in conjunction with pharmacology experiments, in which drugs are applied to block specic channels. Several congurations of this method correspond to whole cell, outside-out and inside-out models (Fig. 3).
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
(a)
Electrode
Whole cell methods continue to be popular for neuropharmacology studies because they allow researchers to understand the complex actions and regulation of receptors gating ion channels.
Outside-out model
In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This conguration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7).
Ion Channel
Inside-out model
In the inside-out model, gigaseal formation is followed by a sharp pull, which detaches the cell membrane and exposes the inner membrane of the cell to the bathing solution. The portion of the membrane that was on the inside of the cell is then on the outside of the pipette, whereas the outer portion is in equilibrium with the uid inside the pipette (Fig. 3c). This conguration is useful for studying the effects of intracellular molecules or drugs on individual ion channels and has been used in pharmacology experiments to study ligand-gated ion channel activity. For example, benzodiazepines, which are used to treat anxiety, are thought to augment synaptic inhibition in the central nervous system. Inside-out patch clamp was used to study the effect of the benzodiazepine, diazepam, on single-channel conductance (8). Diazepam caused a seven-fold increase in the conductance of GABAA chloride channels from rat cultured hippocampal neurons (Fig. 2c). This study demonstrates that the inside-out method can provide information about the effect of drugs on the current of a particular ion channel. Inside-out patch clamp has also been used to study pain sensory transduction in rat ganglion neurons through capsaicin-activated ion channels (9). Capsaicin is known to activate certain cation channels and cause severe pain. One study used patch clamp to determine that 12-(S)-hydroxyeicosatetraenoic acid (HPETE), a lipoxygenase product, was an endogenous activator of capsaicin channels. The inside-out technique is widely used to study various channels, both inhibitory and excitatory, and various preparations from retinal neurons to central neurons. For example, inside-out patch clamp has been used to study the photoresponse of rat intrinsically photosensitive retinal ganglion cells (ipRGCs), which are photoreceptors that control pupil response (10). In this study, inside-out recordings showed that ipRGCs are photosensitive. This study gives insight into the complex cascade of events that lead to vision.
(c)
Outside-out Method
Inside-out Method
Figure 3 Schematic of a) whole cell, b) outside-out, and c) inside-out patch clamp methods.
by the movement of positive ions into the postsynaptic cell through glutamate-mediated channels, were monitored. This study examined whether adenosine receptor activation could modulate EPSCs. Whole cell patch clamp was performed on neurons in striatal rat brain slices. Figure 2b shows an EPSC evoked by electrical stimulation before and after application of an A2A adenosine receptor agonist. The decreased amplitude of the EPSC after the adenosine receptor agonist is applied suggests that adenosine receptor activation inhibits glutamate channel-mediated EPSCs (5). Many researchers also study inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid-(GABA-)-gated chloride channels. For example, whole cell patch clamp was used to differentiate two different types of IPSCs in substantia gelatinosa neurons of the mouse spinal cord (6). IPSCs with a fast decay had a different pharmacological prole than those with a slow decay. 4
Extracellular methods
With intracellular methods, the change in voltage caused by the uctuation of ions across the cell membrane is measured. However, transmembrane ion concentration uxes also generate
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
HP
S1
VPM
Spikes/bin
Figure 4 Electrically evoked single-unit tactile responses recorded with microwire multielectrode arrays in rat hippocampus CA1, primary somatosensory cortex, and ventral posteromedial nucleus brain regions. (Upper) Raster plot of single-unit spikes before and after electrical current stimulation to whiskers. Each row is a separate trial. (Lower) Summed activity for all trials in 1-ms bins that demonstrate a response to electrical stimulation. The graphs show different latencies in ring for the three different regions. [Data reprinted from Ref. (13). Copyright (2007) National Academy of Sciences, U.S.]
an electric eld in the extracellular space, and these changes in electrical activity can be measured by extracellular recordings. Both invasive and noninvasive techniques are routinely used. Extracellular techniques can be used in many preparations, including awake, behaving animals. In a typical invasive setup, a wire electrode or an array of wires is implanted into the brain, and changes in potential are measured. Electrodes that are directly next to a neuron will record voltage changes when that neuron res. This setup has been used to measure neuronal ring patterns in animals performing complex behaviors, such as drug self-administration (11). These studies have shown that dopamine neurons re as predictive signals of reward and will re in response to cues that have been previously associated with rewards (12). Multielectrode arrays have also been used to map the response of different neurons to external stimulations. For example, Nicolelis et al. have used multichannel electrodes to map tactile responses of an anesthetized rat (13). Figure 4 demonstrates the neuronal response to stimulation of the infraorbital nerve, which carries information from the whiskers mechanoreceptors to the brain. Typical data is shown, in which ring patterns before and after the stimulus (dashed line, time 0) are given for multiple trials (top of gures) and then binned to get average ring rates (histograms). The results show that the hippocampal neurons have a longer latency to re but are activated for longer than the neurons in the somatosensory cortex and thalamic ventral posteromedial nucleus. Such studies help identify and interpret the neuronal pathways that process tactile stimulation. In a noninvasive setup, surface electrodes are placed on the surface of the skin and can be used to study individual or multiple neurons. Additionally, many studies look at motor units, which are motor neurons and all muscle bers that the neuron innervates. For example, stable recordings of single motor unit potentials can be made using electromyography (14). Surface electrodes are of interest in electroencephalography (EEG) measurements, in which electrodes are placed on the scalp. Noninvasive EEG measurements are particularly useful in human studies. For example, EEG recordings have been used to study adenosinergic neurotransmission in homeostatic sleepwake regulation in humans (15). These studies found that adenosinergic neurotransmission plays a role in non-rapid-eye-movement sleep homeostasis.
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Microdialysis
Microdialysis has served as a powerful tool in the direct measurement of cerebral neurotransmitters. It can be used as a qualitative technique to monitor changes in neurotransmitters or as a quantitative technique to determine actual neurotransmitter concentrations such as basal levels. Measurements are made using a probe sheathed with a semipermeable membrane, which allows extracellular molecules of a low molecular weight, such as neurotransmitters, to pass but excludes larger molecules such as proteins. The probe is perfused with articial cerebral spinal uid, and molecules diffuse across the membrane according to their concentration gradients. Fluid fractions are collected at the probe outlet. Fractions are typically analyzed using separation techniques, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Microdialysis can provide both highly selective and sensitive analysis of neurochemical changes, but the temporal resolution has been limited traditionally to 520 minutes (21, 22). Continuous sampling of chemical events allows processes such as drug metabolism and neurotransmitter release to be studied. Microdialysis also is useful for studying neurotransmission in various preparations, including in behaving animals.
Microdialysis Theory
In microdialysis, a solution isotonic to that being sampled is perfused through an inlet tube of the probe (Fig. 5). A concentration gradient is established on each side of the membrane between the perfusate and the sampling region. This gradient causes neurochemicals from the sampling region to diffuse through the membrane, and the compounds are collected for analysis at the outlet of the probe. Analyte removal from the sampling region keeps the concentration gradient intact and allows continual sampling of the extracellular space. This process is highly dependent on ow rate of perfusion. At typical ow rates, the concentration in the dialysate, C out , is less than the actual concentration in the extracellular uid, Cext (23). The ratio of Cout /Cext is dened as relative recovery, R, and must be considered for probe calibration and sampling optimization. In vitro , R is easily calculated because the dialysate and the extracellular uid are homogenous; therefore, probe calibration is easily obtained. However, in in vivo studies, calculation of R is difcult because of the active removal of neurotransmitters by uptake and tortuosity. Movement of analytes is impeded by tissue that surrounds the probe, and this movement cannot be easily accounted for with in vitro calibrations. Therefore, the most common method to determine concentrations in vivo is the zero-net ux method, in which known analyte concentrations are added to the perfusate (Cin ), and then the analyte concentration is measured at the probe outlet (C out ). The difference between analyte concentration at the inlet and outlet is used to establish the actual analyte concentration in the tissue, and the relative recovery rate can be calculated. This calibration method can be used to estimate basal levels of neurotransmitters. For example, the zero-net ux method has been used to determine that basal concentrations of dopamine are approximately 13.5 nM (24, 25). Although basal level concentrations 6
Figure 5 Microdialysis probe. Articial cerebral spinal uid is perfused through the inlet, and small molecules can diffuse across the membrane. Fluid is collected at the outlet and analyzed.
of neurotransmitters can be determined with this method, the contribution of tissue damage to the microdialysis signal cannot be neglected. Because microdialysis is an invasive technique, a traumatized layer of tissue will be next to the probe, which inuences the rate of neurotransmitter release and uptake. Bungay et al. have suggested in the case of dopamine that trauma leads to an underestimation of extracellular dopamine concentration and have proposed a quantitative model to correct for that (23).
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250
Sham, N = 4 Placebo + Occlusion N = 12 MgSO4 + Occlusion N = 12
* * 150 * *
* *
100
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0 0 30 60 90 120 150 180 Time Course (min) 210 240 270 300
Figure 6 Effects of intrathecal magnesium sulfate on ischemia-induced release of glutamate in the dialysate collected from the spinal cord of rabbits. Data are expressed as percentage of the mean baseline levels in each group. Aortic artery occlusion to cause ischemia results in an increase in glutamate in the placebo group (open circles), but administration of MgSO4 before ischemia is induced prevents the increase (triangles). A sham group in which ischemia is not induced is also shown (circles). (Data reprinted from Ref. (28) by permission of Lippincott, Williams, and Wilkins.)
Microdialysis studies have played an important role in understanding the relationship between glutamate levels and ischemic events. Glutamate has been shown to increase during a stroke, which contributes directly to neuron damage and additional vulnerability to ischemia (26, 27). Subsequently, methods to reduce glutamate release during an ischemic event have been of interest. Recent microdialysis studies indicate that spinal cord necrosis can be reduced by administering magnesium sulfate to the area experiencing ischemia (28). Figure 6 shows typical data where microdialysis samples were collected and glutamate concentration quantitated every 10 minutes. Intracerebral occlusion of the aortic artery caused ischemia and glutamate levels to increase (open circles). However, when MgSO4 was administered, glutamate did not rise during ischemia, similar to the sham control group. Therefore, microdialysis gives information about how pharmacological treatments could be used to prevent glutamate excitotoxic damage during ischemia. The neurotransmitter dopamine is of particular interest because of the role it plays in brain processes related to movement, cognition, reward, and addiction. Changes in basal levels of dopamine have been monitored using microdialysis to understand the effects of drugs of abuse, such as cocaine, on neurotransmission (29). Dopamine levels have also been measured in different behavioral paradigms. For example, using the zero-net ux method, Chefer et al. have demonstrated an increase in basal dopamine levels in the nucleus accumbens following cocaine stimulation (30). Moreover, they coupled locomotor response to novelty with dopamine levels in response to cocaine. Male rats were screened as either high responders or low responders to environmental stimuli, and high responders showed the greatest increase in dopamine after cocaine. More studies have investigated the effect of drugs, such as cocaine, morphine, and amphetamine, on dopamine levels to determine genetic factors in behavior response and drug abuse (29).
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500 400 % Basal 300 200 100 0 10 0 fox odor 10 20 Time (min) 30 Venton et al. 2006 (CE) Hotsenpiller and Wolf 2003 (HPLC)
Figure 7 Glutamate changes in the nucleus accumbens after presentation of the predator fox odor 2,5-dihydro-2,4,5-trimethylthiazoline. Data are shown as a percentage of basal levels, and presentation of the fox odor causes glutamate to increase. Microdialysis coupled with CE data (solid circles) are compared with previous studies of microdialysis coupled with HPLC (open triangles). Microdialysis coupled to CE provided information not previously found with HPLC because of increased time resolution. (Data adapted from Ref. (31) by permission from Blackwell Publishing.)
electroactive, including dopamine, norepinephrine, epinephrine, and serotonin, and can be detected directly using microelectrodes. The small size of typical microelectrodes, from 7 to 30 m in diameter, results in less tissue damage than the implantation of larger probes, such as microdialysis probes or conventional electrodes. Three of the most common methods of voltammetric detection for neurotransmitters are amperometry, high-speed chronoamperometry, and fast-scan cyclic voltammetry. In each of these methods, a voltage waveform is applied to the electrode. When the potential is sufcient, electroactive neurochemicals can be oxidized or reduced, and a current is measured that is proportional to the concentration of analyte at the electrode surface. The range of voltages that can be used is nite because of the possible oxidation or reduction of physiological solutions and oxygen, which limit detection to the range of about 1 V to 1.5 V. The shape of the potential waveform applied in each technique is unique, and the resulting chemical information is different for each method (41).
Electrochemical Techniques
Electrochemical detection is one of the most common methods for neurotransmitter monitoring. Many neurotransmitters are 8
Chronoamperometry
For high-speed chronoamperometry, a recurrent square waveform is applied to the electrode, with potentials sufcient
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
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(b)
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2.0 Micromolar DA 1.5 1.0 0.5 0.0 0 Nomifensine
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30 pmol DA 30 pmol DA 30 pmol DA 160 320 480 640 800
Time (Seconds)
Figure 8 Electrochemical techniques. Applied potential versus time and resulting current versus time graphs for a) amperometry, b) high-speed chronoamperometry, and c) fast-scan cyclic voltammetry. The dotted circle in b) shows where currents are measured after the charging current has decayed. Panels df show real data from experiments using each of the techniques. Amperometry data (d) show a current increase that corresponds to detection of electroactive species. Each peak is representative of dopamine release from a single vesicle in retinal neurons, and the area under each peak was used to quantitate the number of molecules released. (Data taken from Ref. (43) by permission from the American Chemical Society.) e) Dopamine concentrations measured by chronoamperometry. Upward-facing arrows indicate time of dopamine injection; downward-facing arrows indicate time of nomifensine injection. Curve A is dopamine detected after injection of 30 pmol of DA in normal tissue, whereas traces B and C are after the dopamine uptake inhibitor nomifensine was administered. Uptake rate is reduced by nomifensine as seen by the increased concentration and the slower return to baseline after injection. (Data from Ref. (47) used by permission from Elsevier.) f) Cyclic voltammetry data of dopamine concentrations from cocaine-seeking rats. The cyclic voltammogram (inset) veries that dopamine is being detected. The trace shows the current at the dopamine oxidation potential, converted to concentration based on calibration data. The rst triangle indicates when the rat approaches the lever, and the second shows when it presses the lever to receive an intravenous injection of cocaine. The dopamine increase before the introduction of cocaine shows that one role of dopamine is as an anticipatory signal for reward. (Data reprinted from Ref. (51) with permission from Macmillan Publishers Ltd.)
enough to oxidize the analyte of interest and subsequently reduce the analyte to the initial form. Rapidly changing the potential causes an inherent charging current at the electrode surface, but this current is dissipated quickly because of the micron size of the electrode. Therefore, the current measured at the end of the step, after the charging current has decayed, is proportional to the analyte concentration (Fig. 8b). The ratio of the peak reduction current to the peak oxidation current is a measure of the reversibility of the electrochemical reaction and can be used to differentiate between some molecules. However, this selectivity is limited, and positively identifying the analyte solely by this technique is difcult. The chronoamperometry waveform is typically repeated at 525 Hz. Greg Gerhardt pioneered a chronoamperometry method that has been used to observe diffusion and uptake parameters in the rat brain (45). In this technique an exogenous neurotransmitter, such as dopamine, is introduced into the brain by pressure ejection or iontophoresis. The microelectrode is a known distance from the ejection pipette, so the analyte concentration detected at the microelectrode is a function of extracellular diffusion and uptake by the dopamine transporter. Because an exogenous neurotransmitter is introduced, the analyte being detected is known, and selectivity is not a problem. After a dopamine
ejection, Gerhardts group could compare dopamine uptake rates of wild-type rats and rats that had been treated with nomifensine, a dopamine uptake inhibitor (46, 47). They found that nomifensine slowed dopamine uptake rates, as seen by the slower return to baseline in Fig. 8e, and that uptake inhibition increased the concentration of dopamine detected at the electrode. This study was extended to compare dopamine uptake rates with rats that had their transporters disabled during development (47, 48). Recently, chronoamperometry has also been used to show that mice lacking brain-derived neurotrophic factor have slower serotonin clearance caused by impaired serotonin transporter function (49).
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the charging current is relatively stable at carbon electrodes, the current before analyte introduction can be subtracted from the current after analyte introduction. This method is known as background-subtracted FSCV, and it is best used for measuring concentration changes. The data collected is a plot of current versus voltage, called a cyclic voltammogram, which gives a chemical signature of the molecule detected. The peak locations and shapes help differentiate between different molecules, which gives this method the most selectivity of any of the electrochemical techniques discussed here. Some molecules, such as dopamine and norepinephrine, however, have similar CVs and are difcult to resolve. R. Mark Wightmans lab used carbon-ber microelectrodes to pioneer the use of FSCV in measuring dopamine concentration changes with high temporal resolution. Early experiments focused on measuring dopamine concentration changes after electrical stimulation of the dopamine cell bodies (50). In more recent studies, behaviorally evoked dopamine concentrations have been measured. One example is the detection of dopamine in rats when cocaine was self-administered (Fig. 8f) (51). The subsecond temporal resolution allowed the observation that extracellular dopamine increased continuously for about 4 seconds before the lever press for cocaine administration and after the lever press. This observation shows that one role of dopamine is as an anticipatory signal (51). FSCV has also been used to measure other neurotransmitters, such as serotonin (52) and norepinephrine (53), in vivo and in brain slices. Our lab has recently extended the use of FSCV to detect the neuromodulator adenosine successfully. In vivo detection of adenosine and dopamine simultaneously was achieved, and the temporal resolution allowed us to demonstrate that adenosine accumulation was slower than dopamine (38, 54). Fast-scan cyclic voltammetry is the best choice for detecting neurotransmitters that undergo volume transmission in vivo because the cyclic voltammogram provides a means to identify the species detected and the high temporal resolution allows correlation with behavior.
References
1. Staal RG, Mosharov EV, Sulzer D. Dopamine neurons release transmitter via a ickering fusion pore. Nat. Neurosci. 2004;7:341346. Smith CUM. Elements of Molecular Neurobiology, 2nd edition. 1996. Wiley, New York. Zoli M, Torri C, Ferrari R, Jansson A, Zini I, Fuxe K, Agnati LF. The emergence of the volume transmission concept. Brain Res. Rev. 1998;26:136147.
2. 3.
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
4.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
15. 16.
17.
18. 19.
20.
21. 22.
23.
Verma S, Robertson AP, Martin RJ. The nematode neuropeptide, AF2 (KHEYLRF-NH2), increases voltage-activated calcium currents in Ascaris suum muscle. Br. J. Pharmacol. 2007;151: 888899. Gerevich Z, Wirkner K, Illes P. Adenosine A(2A) receptors inhibit the N-methyl-D-aspartate component of excitatory synaptic currents in rat striatal neurons. Eur. J. Pharmacol. 2002; 451:161164. Takahashi A, Tokunaga A, Yamanaka H, Mashimo T, Noguchi K, Uchida I. Two types of GABAergic miniature inhibitory postsynaptic currents in mouse substantia gelatinosa neurons. Eur. J. Pharmacol. 2006;553:120128. Jardemark K, Orwar O, Jacobson I, Moscho A, Zare RN. Patch clamp detection in capillary electrophoresis. Anal. Chem. 1997;69:34273434. Eghbali M, Curmi JP, Birnir B, Gage PW. Hippocampal GABA(A) channel conductance increased ky diazepam. Nature. 1997;388: 7175. Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D, Oh U. Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc. Natl. Acad. Sci. USA. 2000;97:61556160. Graham DM, Wong KY, Shapiro P, Frederick C, Pattabiraman K, Berson DM. Melanopsin ganglion cells use a membrane-associated rhabdomeric phototransduction cascade. J. Neurophys. 2008;99: 25222532. Song D, Chan RHM, Marmarelis VZ, Hampson RE, Deadwyler SA, Berger TW. Nonlinear dynamic modeling of spike train transformations for hippocampal-cortical prostheses. IEEE. T. Bio-med. Eng. 2007;54:10531066. Schultz W. Behavioral dopamine signals. Trends Neurosci. 2007; 30:203210. Pereira A, Ribeiro S, Wiest M, Moore LC, Pantoja J, Lin SC, Nicolelis MA. Processing of tactile information by the hippocampus. Proc. Natl. Acad. Sci. U.S.A. 2007;104:1828618291. Christova L, Stephanova D, Kossev A. Branched EMG electrodes for stable and selective recording of single motor unit potentials in humans. Biomedizinische Technik. 2007;52:117121. Landolt HP. Sleep homeostasis: A role for adenosine in humans? Biochem Pharmacol. 2008;75:20702079. Hamill OP, McBride DW. Induced membrane hypo/hypermechanosensitivity: A limitation of patch-clamp recording. Annu. Rev. Physiol. 1997;59:621631. Fertig N, Blick RH, Behrends JC. Whole cell patch clamp recording performed on a planar glass chip. Biophys. J. 2002;82:3056 3062. Priest BT, Swensen AM, McManus OB. Automated electrophysiology in drug discovery. Curr. Pharm. Design 2007;13:23252337. Semmler JG, Kornatz KW, Dinenno DV, Zhou S, Enoka RM. Motor unit synchronisation is enhanced during slow lengthening contractions of a hand muscle. J. Phys. 2002;545:681695. Brown EN, Kass RE, Mitra PP. Multiple neural spike train data analysis: state-of-the-art and future challenges. Nat. Neurosci. 2004;7:456461. Chaurasia CS. In vivo microdialysis sampling: theory and applications. Biomed. Chromatogr. 1999;13:317332. Jing FC, Chen H, Li CL. Rapid determination of dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection. Biomed. Envi. Sci. 2007;20:317320. Bungay PM, Newton-Vinson P, Isele W, Garris PA, Justice JB. Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma. J. Neurochem. 2003;86: 932946.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Tang A, Bungay PM, Gonzales RA. Characterization of probe and tissue factors that inuence interpretation of quantitative microdialysis experiments for dopamine. J. Neurosci. Methods 2003;126:111. Parsons LH, Smith AD, Justice J. The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens. J. Neurosci. Meth. 1991;40:139147. Cui YL, Zhang L, Utsunomiya K, Yanase H, Mitani A, Kataoka K. Ischemia-induced glutamate release in the dentate gyrus. A microdialysis study in the gerbil. Neurosci. Lett. 1999;271:191194. Benveniste H, Drejer J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral-ischemia monitored by intracerebral microdialysis. J. Neurochem. 1984;43:13691374. Jellish WS, Zhang X, Langen KE, Spector MS, Scalfani MT, White FA. Intrathecal magnesium sulfate administration at the time of experimental ischemia improves neurological functioning by reducing acute and delayed loss of motor neurons in the spinal cord. Anesthesiology. 2008;108:7886. Cadoni C, Di Chiara G. Differences in dopamine responsiveness to drugs of abuse in the nucleus accurnbens shell and core of Lewis and Fischer 344 rats. J. Neurochem. 2007;103:487499. Chefer VI, Zakharova I, Shippenberg TS. Enhanced responsiveness to novelty and cocaine is associated with decreased basal dopamine uptake and release in the nucleus accumbens: Quantitative microdialysis in rats under transient conditions. J. Neurosci. 2003;23:30763084. Venton BJ, Robinson TE, Kennedy RT. Transient changes in nucleus accumbens amino acid concentrations correlate with individual responsivity to the predator fox odor 2,5-dihydro-2,4,5-trimethylthiazoline. J. Neurochem. 2006;96:236246. Hotsenpiller G, Wolf ME. Baclofen attenuates conditioned locomotion to cues associated with cocaine administration and stabilized extracellular glutamate levels in the rat nucleus accumbens. Neurosci. 2003;118:123134. Venton BJ, Robinson TE, Kennedy RT, Maren S. Dynamic amino acid increases in the basolateral amygdala during acquisition and expression of conditioned fear. Eur. J. Neurosci. 2006;23:33913398. Lena I, Parrot S, Deschaux O, Muffat-Joly S, Sauvinet V, Renaud B, Suaud-Chagny MF, Gottesmann C. Variations in extracellular levels of dopamine, noradrenaline, glutamate, and aspartate across the sleep-wake cycle in the medial prefrontal cortex and nucleus accumbens of freely moving rats. J. Neurosci. Res. 2005;81:891899. Cellar NA, Burns ST, Meiners JC, Chen H, Kennedy RT. Microuidic chip for low-ow push-pull perfusion sampling in vivo with on-line analysis of amino acids. Anal. Chem. 2005;77:70677073. Khan AS, Michael AC. Invasive consequences of using microelectrodes and microdialysis probes in the brain. TrAC. Trends Anal. Chem. 2003;22:503508. Clapp-Lilly KL, Roberts RC, Duffy LK, Irons KP, Hu Y, Drew KL. An ultrastructural analysis of tissue surrounding a microdialysis probe. J. Neurosci. Methods. 1999;90:129142. Swamy BEK, Venton BJ. Susbsecond detection of physiological adenosine concentrations using fast-scan cyclic voltammetry. Anal. Chem. 2007;79:744750. Crespi F, Mobius C. In vivo selective monitoring of basal levels of cerebral dopamine using voltammetry with Naon modied (NA-CRO) carbon bre micro-electrodes. J. Neurosci. Methods 1992;42:149161.
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
11
40.
41.
42.
43.
44. 45.
46.
47.
48.
49.
50.
51.
52.
53.
54. 55.
56.
57.
Parkin MC, Hopwood SE, Jones DA, Hashemi P, Landolt H, Fabricius M, Lauritzen M, Boutelle MG, Strong AJ. Dynamic changes in brain glucose and lactate in pericontusional areas of the human cerebral cortex, monitored with rapid sampling on-line microdialysis: relationship with depolarisation-like events. J. Cereb. Blood Flow. 2005;25:402413. Michael DJ, Wightman RM. Electrochemical monitoring of biogenic amine neurotransmission in real time. J. Pharm. Biomed. Anal. 1999;19:3346. Leszczyszyn DJ, Jankowski JA, Viveros OH, Diliberto EJ Jr., Near JA, Wightman RM. Nicotinic receptor-mediated catecholamine secretion from individual chromafn cells. Chemical evidence for exocytosis. J. Biol. Chem. 1990;265:1473614737. Hochstetler SE, Puopolo M, Gustincich S, Raviola E, Wightman RM. Real-time amperometric measurements of zeptomole quantities of dopamine released from neurons. Anal. Chem. 2000;72:489496. Wightman RM, Haynes CL. Synaptic vesicles really do kiss and run. Nat. Neurosci. 2004;7:321322. Zahniser NR, Dickinson SD, Gerhardt GA. High-speed chronoamperometric electrochemical measurements of dopamine clearance. Methods Enzymol. 1998;296:708719. Costall B, Kelly DM, Naylor RJ. Nomifensine: a potent dopaminergic agonist of antiparkinson potential. Psychopharmacologia 1975;41:153164. Luthman J, Friedemann MN, Hoffer BJ, Gerhardt GA. In vivo electrochemical measurements of exogenous dopamine clearance in normal and neonatal 6-hydroxydopamine-treated rat striatum. Exp. Neurol. 1993;122:273282. Cass WA, Zahniser NR, Flach KA, Gerhardt GA. Clearance of exogenous dopamine in rat dorsal striatum and nucleus accumbens: role of metabolism and effects of locally applied uptake inhibitors. J. Neurochem. 1993;61:22692278. Daws LC, Munn JL, Valdez MF, Frosto-Burke T, Hensler JG. Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): in vivo studies in BDNF-decient mice. J. Neurochem. 2007;101:641651. Robinson DL, Venton BJ, Heien MLAV, Wightman RM. Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo. Clin. Chem. 2003;49:17631773. Phillips PEM, Stuber GD, Heien ML, Wightman RM, Carelli RM. Subsecond dopamine release triggers cocaine seeking. Nature. 2003;422:614618. Bunin MA, Wightman RM. Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J. Neurosci. 1998;18:4854 4860. Miles PR, Mundorf ML, Wightman RM. Release and uptake of catecholamines in the bed nucleus of the stria terminalis measured in the mouse brain slice. Synapse. 2002;44:188197. Cechova S, Venton BJ. Transient adenosine efux in the rat caudate-putamen. J. Neurochem. 2008;105:12531263. Kulagina NV, Michael AC. Monitoring hydrogen peroxide in the extracellular space of the brain with amperometric microsensors. Anal. Chem. 2003;75:48754881. Oldenziel WH, Dijkstra G, Cremers TIFH, Westerink BHC. In vivo monitoring of extracellular glutamate in the brain with a microsensor. Brain Res. 2006;1118:3442. Cheer JF, Aragona BJ, Heien MLAV, Seipel AT, Carelli RM, Wightman RM. Coordinated accumbal dopamine release and neural activity drive goal-directed behavior. Neuron. 2007;54: 237244.
58.
59.
60.
61.
62.
Logothetis NK, Pauls J, Augath M, Trinath T, Oeltermann A. Neurophysiological investigation of the basis of the fMRI signal. Nature. 2001;412:150157. Pappata S, Salvatore E, Postiglione A. In vivo imaging of neurotransmission and brain receptors in dementia. J. Neuroimaging. 2008;18:111124. Lohse MJ, Bunemann M, Hoffmann C, Vilardaga JP, Nikolaev VO. Monitoring receptor signaling by intramolecular FRET. Curr. Opin. Pharmacol. 2007;7:547553. Garris PA, Ensman R, Poehlman J, Alexander A, Langley PE, Sandberg SG, Greco PG, Wightman RM, Rebec GV. Wireless transmission of fast-scan cyclic voltammetry at a carbon-ber microelectrode: proof of principle. J. Neurosci. Methods. 2004;140: 103115. Schregardus DS, Pieneman AW, Ter Maat A, Jansen RF, Brouwer TJF, Gahr ML. A lightweight telemetry system for recording neuronal activity in freely behaving small animals. J. Neurosci. Methods. 2006;155:6271.
Further Reading
Zigmond MJ, Bloom FE, Landis SC, Roberts JL, Squire LR. Fundamental Neuroscience. 1999. Academic Press, New York. Johnston D, Wu M. Foundations of Cellular Neurophysiology. 1995. Bradford Books, London, UK. Joukhadar C, Muller M. Microdialysis: Current Applications in Clinical Pharmokinetic Studies and its Potential Role in the Future. Clinical Pharmacokinetics 2005;44:895913. Michael AC, Borland LM. Electrochemical Methods for Neuroscience. 2007. CRC Press, New York.
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