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Fast differential ramp voltammetry: a new voltammetric technique designed specifically for use in neuronal tissue
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J. Electroanal. Chem., 282 (1990) 33-49 Elsevier Sequoia S.A., Lausarme - Printed
in The Netherlands
Fast differential ramp voltammetry: a new voltammetric technique designed specifically for use in neuronal tissue J. Millar and G.V. Williams Dept. of Physiology, (Received
Turner Street, London El 2AD (Great Britain)
13 July 1989; in revised form 13 November
1989)
ABSTRACT This paper describes details of a new voltammetric technique designed specifically for use in living, and the function of the especially neuronal, tissue. Neuronal tissue is an “active” electrical environment, nerve cells may be disrupted by some voltammetric procedures. The new technique, which is called “Fast Differential Ramp Voltammetry (FDRV)“, has been developed from Fast Cyclic Voltammetry (FCV). FCV can be shown not to interfere with ongoing neuronal activity because the working electrode can be switched to record the electrical activity of the surrounding nerve cells in between voltammetric scans. The charging currents generated in FCV are however affected by local neuronal activity, and in the past this has limited the resolution of FCV in viva. In FDRV these effects are reduced by a double or quadruple offset scan technique which cancels out most of the variation in signal due to fluctuations in charging currents. The resolution of FDRV should be adequate to study “basal” levels of neurotransmitters in viva. The technique is immune to electrocatalytic effects and can resolve separate peaks for mixtures of re-reducible and non re-reducible electroactive compounds. This enables it to be used for separate measurement of, for example, dopamine and ascorbic acid in mixtures of the two.
INTRODUCTION
The last decade has seen the development of voltammetry at carbon fibre electrodes into an important tool for the investigation of neurotransmitter function in the mammalian brain (see refs. l-4 for reviews). This development has largely stemmed from the work of Ralph Adams and his colleagues in the early 1970’s, who first applied voltammetric methods to the study of neurotransmitter release in vivo [5-S]. A fundamental characteristic of voltammetry is that the analysis requires the application of a voltage waveform to the tissue being studied. In particular, many voltammetric techniques use voltage pulses as part of the analytical waveform. These include chronoamperometry [9] normal pulse voltammetry [lO,ll] differential pulse voltammetry [12], and differential double pulse voltammetry [12,13]. However, 0022-0728/90/$03.50
0 1990 Elsevier Sequoia
S.A.
34
voltage pulses are normally used by neurobiologists as a means of st~ulating nerve cells, and it is known that currents of less than 1 pA can activate a cell under the right conditions [14-161. Thus, before most neurobiologists will accept the validity of in-vivo voltammetric data, they will need to be convinced that the currents flowing through the working and auxiliary electrodes do not affect the normal functio~ng of the tissue. In other words, the applied waveforms must be shown unequivocally not to stimulate or block the activity of nerve cells. The cells must be recorded as close to the working electrode as possible, for that is where the current density will be greatest; it is not adequate to show that cells at a distance from the working electrode are unaffected, although if distant cells were affected, obviously the technique would be unacceptable. A second major consideration for in-vivo volt~et~ is the fact that the brain is an “active” electrical environment, in the sense that the neurones and glial cells produce ac and dc voltages as part of their normal function. There may also be spontaneous changes in tissue impedance due to the activity of the cells [17]. Thus a second requirement for a voltammetric technique to be used in vivo is that it must allow for and accommodate the normal fluctuations in potential and impedance found in living tissue. The third and final consideration will involve the size of the electrodes that are used. The diameters of the cell bodies of nerve cells lie mostly in the range of 10 to 50 pm. These cells are packed closely together with their supporting glial cells such that the extracellular space in neuronal tissue is normally no more than about lo-20% Any electrode inserted into this tissue will cause damage, but if the electrode diameter can be made comparable to or smaller than the typical cell diameter, then it can be hoped that the damage will be confined to the cells directly in the path of the electrode. Larger electrodes will not only destroy the cells directly in their path, but will also inflict “crush injuries” on cells lateral to the electrode. In this paper we describe fast differential ramp voltammetry (FDRV), using 8 pm diameter carbon fibre microelectrodes. This technique has been developed from fast cyclic voltammetry (FCV) 118,191,specifically as an attempt to satisfy the conditions set down above. A special feature of FDRV is the elimination of electrocatalytic effects in the analysis without alteration of normal brain levels of electroactive materials, and thus the possibility of simultaneous independent quantification of catecholamine and ascorbic acid in mixtures of the two. EXPERIMENTAL
Electrodes and potentiostat
configuration
A four-electrode system is used, comprising a working, auxiliary, and two reference electrodes. The two reference electrodes are designated micro-reference and macro-reference. The working and micro-reference electrodes are made from carbon fibres, incorporated together into a compound glass microelectrode. The construction of this compound carbon fibre electrode is similar to that previously described for single electrodes [20], except that the glass blank is a theta or “quad”
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I
I 20 pm
Fig. 1. Appearance of the tip region of a twin carbon fibre microelectrode. The glass insulation is on the left, with the exposed fibres, cut or spark etched to the appropriate length, protruding on the right. (A) Working electrode; (B) micro-reference electrode.
type. (Theta glass tubing has a septum down the middle so that in cross-section it looks like the Greek letter theta; two microelectrode “barrels” can thus be obtained from one piece of glass when it is pulled into a microelectrode. Quad or quadrant glass contains a septum that that is cruciform in cross-section and thus divides the internal space of the tube into four sections, like a pie chart. This can be pulled to make a four-barrel electrode.) Single carbon fibres are inserted into two compartments in the tube, which can then be pulled in a microelectrode puller to leave the two fibres protruding from two separate barrels beyond the glass at the tip. The fibres are either cut mechanically or etched with a high-voltage spark [21] to a tip length of 20 to 50 pm, but receive no other “pre-treatment” or coating. Contact with the fibres in the blunt end of the electrode can be made with a fine copper wire dipped in silver-doped epoxy glue. Figure 1 shows the appearance of the compound electrode tip in a two-barrel electrode. The macro-reference electrode is a conventional silver/ silver chloride electrode and the auxiliary electrode (which may in theory be of any conducting material) is in practice usually either another Ag/AgCl electrode or a stainless steel wire. The micro-reference is capacitatively coupled through a first-order high-pass filter to the reference circuit. The macro-reference electrode should be placed on the surface of the brain, or at some other point where high frequency changes in potential do not occur. A total reference signal is then synthesised from the sum of the macro-reference signal and the high-pass filtered micro-reference signal. In order to obtain extracellular voltage recordings in the intervals between voltammetric scan epochs, and to enable multiple working electrodes to be used asynchronously in the same preparation, the usual voltammetric system of an active auxiliary electrode and a passive working electrode is reversed in FDRV. The auxiliary electrode is permanently connected to ground (earth) potential, while the driving voltage waveform is applied to the working electrode. We call this the “grounded auxiliary system”. The total potentiostat using this grounded auxiliary system and the compound reference circuit is shown in Fig. 2. Voltammetric scan parameters; the “time share” recording system This is identical in principle to that used for FCV. The working electrode is connected to the voltammetric amplifier for only 40 ms at a time. In between these
I
Logic Drive to Analog Gate
Voltammetry
1 Micro-reference
OUtPUt
Electrode
Jp!G
Macro-reference
160K
47 M
J..> i
\
Preparatlon
u
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Fig. 2. The four-electrode potentiostat circuit for Fast Differential Ramp Voltammetry (FDRV). Analog gate: l/2 DG303 or similar. Operational amplifiers: (1, 2, 5, 7) OPAlll; (3, 4, 6, 8) 0PA2111 or similar. Resistors: 0.1% metal film (1% for 1 and 47 I@.
40 ms “scan epochs” the working electrode is switched electronically to the input of a voltage-follower amplifier. The voltage signal obtained from this follower can then be amplified and filtered to give a record of the ongoing electrical activity of the nerve cells in the intervals between voltammetric scan epochs [18]. By recording the electrical activity of the nerve cells immediately before and after each voltammetric scan epoch it can be seen whether or not the electrochemical analysis has affected the spontaneous activity of the neurones. In our present studies, the scan epochs are repeated at intervals of 250 or 500 ms. Thus at 500 ms intervals, one obtain 450 ms periods of voltage recording (500 ms between epochs minus 10 ms “settling time”) alternating with 40 ms of voltammetric recordings. Fast Differential Ramp Voltammetry (FDR V) FDRV uses fast triangular waves (“ramps”) to oxidise and reduce the electroactive material in solution. These waves are biphasic, positive-negative going waveforms. FCV uses a triphasic waveform, but the initial negative-going part of the FCV wave does not seem to be necessary and may be discarded. A single biphasic wave can, for the sake of clarity, be referred to as a “scan”. FDRV can be carried out in two ways: single or dual mode. In single FDRV a pair of scans is generated in each scan epoch (Fig. 3A). The scan in the second 20 ms is an exact repeat of the scan in the first 20 ms, but offset negative by a fixed voltage step, usually 200 mV. The amplitude of the positive and negative peaks within the scan are variable (by an
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Fig. 3. Drive waveforms and background signals in FDRV. (A) Twin voltage ramp patterns (scans) used in single model FDRV. (B) Background current generated by these waveforms when a carbon fibre working electrode is immersed in phosphate-buffers saline (PBS). (C) The two waveforms in (B) electronically subtracted to give a “~fferenti~ signal. (D) A ~fferenti~ signal with a earher “standard” differential signal subtracted from it; this is the “subtracted differential” or SD signal. (E) SD signal from (D) at x 50 gain. Calibration bar: (A) 400 mV, (B, C, D) 100 nA, (E) 2 nA.
analog amplifier) but the duration is always 10 ms. Thus the rate of change of voltage, dV/db, varies as a function of the ~p~t~de. For an ~p~tude of f 1 V, dV/dt would be 400 V/s. In practice, a positive peak of f 900 mV, and a negative peak of - 300 mV has been found suitable for detection of most catecholamines (in later experiments, some improvement in detection in vivo was found with scans to -t 1.4 V instead of 900 mv). Figure 3A shows a pair of voltage scans generated in one scan epoch. The corresponding background currents generated in the carbon fibre working electrode by these voltages are shown underneath, in Fig. 3B. The working electrodes were immersed in phosphate-buffered 0.9% saline (PBS) at room temperature. These background currents are due to the electrical properties of the double layer at the electrode/electrolyte interface, and are extremely stable at a fixed temperature and pH. The current waveform through the working electrode is converted to a voltage by an operational amplifier used as a current-to-voltage converter (1 in Fig. 2) then amplified to a s&able level (approx. &5 V), digit&d with a precision 12-bit ~~og-to-~~t~ converter and stored el~tro~c~ly. The signal produced during the scan in the second half of the scan epoch is then subtracted digitally point-by-point from the signal produced during the first scan to give a “differential” signal (Fig. 3C). As can be seen, this procedure cancels out most of the backwood signal. The differential signal is then in turn stored and forms a standard or “reference” differential signal. The standard differential signal is subtracted digitally from the differential signal generated in the following scan epoch to give a “subtracted differential” (SD) signal. If no changes have occurred in the solution between the acquisition of the standard and present differential signals, then the SD signal is a
Fig. 4. Background signals from an FDRV scan with and without micro-reference compensation. (A) Drive waveform. (B) Signals in PBS. (C) Signals in rat caudate nucleus. (D) Signals in rat corpus callosum.
straight line (Fig. 3D). This two-stage subtraction process eliminates the background current, but allows faradaic signals to show through, although in an unusual form (see below). To illustrate the noise level of the system, Fig. 3E shows the SD signal at X 50 gain. The noise current was found to depend on tip size; most electrodes generated a noise (peak to peak, 0 to 5 KHz) of between 100 and 1000 pA. The importance of the micro-reference in-vivo electrode can be illustrated by its effect on the background signal. Figure 4 shows the background signals from a typical dual carbon fibre electrode as illustrated in Fig. 1. The voltage scan (Fig. 4A) was + 900 to - 300 mV in 5 ms. Figure 4B shows two superimposed traces obtained in PBS, one with and one without the micro-reference connected. The signal with the micro-reference in circuit is about 5% greater, implying that the impedance of the electrolyte between the working and auxiliary electrodes is a significant fraction of the total impedance. Figure 4C shows the backgrounds from the same electrode with and without the micro-reference with the electrode in the caudate nucleus of an anaesthetised rat; the difference is similar to that seen in vitro. Figure 4D shows the signal with the electrode in the corpus callosum. In this region, the high density of myelinated axons in the neuropil increases the tissue impedance and the signal is considerably attenuated without the micro-reference in circuit. Even in regions
where the impedance is low, the micro-reference is able to compensate for changes in impedance due to neuronal activity, and thus stabilise the charging current signal. Addition of electroactive materials to the solution around the working electrode will generate faradaic oxidation and reduction signals. This is illus~ated in Fig. 5. Figure 5A shows a pair of scan voltages as in Fig. 3A. Figure 5B shows two superimposed current traces obtained first in PBS and then with 20 pm dopamine (DA) present in the PBS. An increase in signal current is visible at two regions; firstly at a positive-going (outsing) voltage and then at a negative-gong (reducing)
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Fig. 5. Faradaic currents from FDRV scans. (A) Drive waveforms. (B) Electrode current before and after addition of dopamine (DA} to give 20 pM final concentration. (C) The two differential signals obtained from the traces in (B). (D) A standard differential signal from the difference between the two signals in (C). (E) SD signal in {Dj at ten times gain. Calibration bar: (A) 400 mV, (B, C, D) 100 nA, (E) 10 ILL.
one. Figure 5C shows the appearance of the differential signal before and after addition of dopamine and Fig. SD shows the corresponding SD signal (i.e. the difference between the two signals in Fig. 32). Finally, Fig. 5E shows the SD signal at X 10 gain. In Figs. 5D and E both the faradaic oxidation wave and the reduction wave have a biphasic, “pseudo-differentiated” appearance. The reason for the biphasic appearance of the faradaic oxidation and reduction waves in the SD signal is that the the signal in the second half of the epoch is generated from a negative-offset form of the drive waveform; since this waveform is a positive-going ramp, it reaches the oxidation potential for dopamine later (relative to the start of the ramp) than in the first scan. In Fig. 53, the dopamine oxidation signal can be seen to be displaced on the background in the second scan as compared to the first scan. When the signals in the first and second halves of the epoch are subtracted, the backgrounds are s~chro~sed and tend to cancel out but the faradaic signals are time-shifted and so the monophasic oxidation and reduction waves are converted to a biphasic, ‘“pseudo-differentiated” form.
Single and dual mode FDRV differ only in the pattern and timecourse of the voltammetric scans used in the 40 ms scan epoch. In dual FDRV, four biphasic scans with faster rise times are used, each one lasting 5 ms, with the last pair offset negative from the first pair. Thus, two subtracted differential signals are obtained
Fig. 6. Dual mode FDRV. (A, B) Voltage and current waveforms similar to Fig. 4, but with two scans at each baseline voltage. (C, D) Differential and standard differential signals for 50 pM DA in PBS. (E) SD signal from (D) at X 10 gain. Calibration: (A) 400 mV, (B, C, D) 200 nA, (E) 20 nA.
per scan epoch, as shown in Fig. 6 (Figs. 5 and 6 both show the signal from 20 pm DA with the same scan amplitudes and offset). In mixtures of electroactive materials or in vivo, dual FDRV has several advantages over single FDRV, which are discussed below. Data collection and analysis
The SD signal is generated once (single mode) or twice (dual mode) per scan epoch in FDRV, at scan epoch rates of up to 16 SK’. This signal may be stored on tape if a wide-bandwidth tape recorder is available, but in fact much of the information in the trace is redundant if a only single electroactive species is changing in concentration at any one time. In this case a reduced data collection system may be used, where the amplitude of the faradaic oxidation or reduction wave is sampled at a fixed voltage on a single scan in each epoch, and this voltage is held between scans by means of a sample-and-hold circuit. This form of data sampling has been used extensively in studies using FCV [22]. A considerable improvement in signal-to-noise ratio is possible in FDRV if two S&H circuits are used to sample the peak and trough of the (biphasic) oxidation wave. The signals from the two circuits are fed into a difference amplifier to give the peak-to-peak amplitude of the SD oxidation wave. This procedure nearly doubles the absolute sensitivity of the sample, and, more importantly, cancels out any low frequency drift
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Fig. 7. Comparison of backwood-subtracts FCV (A) and subtracted differential FDRV (B) signals for 50 nM dopamine in vitro. Dots mark where signals are sampled by sample-and-bold circuits to provide continuous outputs to a chart recorder. (C) Drive waveform for both (A) and (B) signals.
in the sampled signal. Figure 7 shows a comparison of two 50 nM dopamine signals. Figure 7A shows an FCV scan with the background subtracted to show the faradaic oxidation and reduction waves generated by the scan voltage in Fig. 7C. This signal rides on a backwood noise level which can be estimated by subtracting the backgronnd from itself; this is shown as the nearly flat line in Fig. 7A. A single S&H circuit would be most sensitive if sampling at the point shown by the dot, but low fr~uency drift means that the signal-to-noise ratio is only about two to one_ Figure 7B shows the same concentration analysed with FDRV and using a dual S&H circuit at the points shown. Baseline drift is now minimal and the signal to noise ratio is now about five to one. RESULTS
In st~d~d ~o~~ti~ns in vitro the sensitivity of FDRV for most electrodes was found to lie in the region of 10 nM for dopamine. This was the concentration that would produce a signal at the oxidation peak current of approximately double the noise level. Occasionally electrodes were made that could detect less than 5 nM dopamine; these electrodes had very low noise levels; the reason for this has not yet been established. “Standard conditions” can be defined as PBS which was continually stirred by air bubbled through it, m~ntained at a fixed temperature between 15 and 20” C, with the test electrode having stabilised its background by continuous scans at 2 Hz or faster for at least 10 mm before testing. All tests were done with scan epochs repeated at 2 Hz. No ascorbate or other reducing agents were added to the solution. Figure 8A shows the voltage scan for the fourth of a dual FDRV set. Figure 8B shows the SD signal produced on this scan by the addition of 10 nM dopamine to the test solution. Six trials were carried out on a single electrode, washing out the beaker and electrode between trials and allowing the background to stabilise for 5 min before addition of dopamine. The signals 10 s after addition of
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Fig. 8. In-vitro FDRV detection thresholds. (A) Drive waveform. (B) SD signals for 10 nM dopamine in PBS; six traces supe~m~sed. (C) Dual S&H records for a series of additions of 10 nM increments of dopamine to PBS (each addition produces a signal similar to that shown in (B)). (D) Similar data to (C) with a different electrode. Time calibration (A, B) 1 ms, (C, D) 2 min.
dopamine (to allow for mixing) are shown superimposed. A dual sample-and-hold circuit was used to capture the amplitude of the signals at the times marked by dots. Figures 8C and D show the dual S&H signals obtained from two further electrodes. Dopamine was added at the points marked by dots to give a series of IO nM increments in concentration. Each increment of dop~ne can be seen to produce a clearly visible increase in the sampled signal level. In vivo, the ongoing electrical and chemical activity of the nerve cells would be expected to increase the background noise and thus decrease the detectabi~ty of amines. However, when investigating dopamine release in the rat striatum following electrical stimulation of the nigro-striatal pathway [22-241, we have detected changes of less than 20 nM dopamine [21]. Thus FDRV appears to have sufficient sensitivity to detect basal levels of catecholamines in the mammalian brain, which appear to be of the order of 50 nM [25]. Selectivity One of the major problems with many voltammetric techniques is the difficulty of identifying the individual components in a mixture of electroactive materials. In particular, the phenomenon of “electrocatalysis” [26-281 can cause non-linear summation or multiplication of oxidation signals in mixtures of catecholamines and ascorbic acid. This happens when a molecule of catecholamine is re-reduced by a molecule of ascorbate immediately after electro-oxidation at the working electrode. The re-reduced catecholamine may then be oxidised again on the electrode, and a cycle of reactions set up, with the net result that the ascorbate effectively amplifies the dopamine signal, or at least produces a non-linear addition of ascorbate and
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Fig. 9. Absence of electrocatalytic effects in dual FDRV. (A) Dual FDRV voltage scans. (B) SD signals for 1 pM DA in PBS. (C) Similar signals for 1 pM DA in PBS containing 200 pM ascorbic acid (AA). (0) SD signal for 200 pM AA in PBS. (E) SD signal for 200 pM AA in PBS containing 1 pM DA. (F) SD signals (with a new electrode) for a mixture of 200 FM AA and 1 pM DA relative to PBS. (G) Result of subtraction of the second SD signal in (F) from the first.
dopamine signals. Electrocatalytic effects are significant in slow-scan cyclic voltammetry, but get smaller with faster scan rates, because there is a shorter total time allowed for oxidation. In normal FCV electrocatalytic effects have been found to be small, and the faradaic signal produced by a mixture of ascorbate and dopamine is approximately the sum of the individual signals generated by separate solutions (unpublished results). The higher speed of the scans in FDRV as compared to FCV is sufficient to eliminate electrocatalytic effects completely. It has been calculated [27] that the quinone initial product of dopamine electro-oxidation has a half-life of 2.1 ms in the presence of ascorbate. To obtain electrocatalysis, the quinone must be reduced and then the dopamine re-oxidised. Inspection of dual FDRV traces shows that the dopamine oxidation takes about 2 ms. Thus a reasonable minimum time for a cycle of electrocatalysis to occur would be about 6 ms. However, since the total time for oxidation in dual FDRV is less than 4 ms, there is insufficient time for electrocatalysis to occur, and dopamine and ascorbate signals sum linearly. Figure 9 illustrates this effect. Figure 9A shows the last pair of voltage scans in an FDRV set; Fig. 9B shows the SD signals for 1 PM DA in PBS. Figure 9C shows similar data for the same electrode with the same concentration of DA but this time added to PBS already containing 200 PM ascorbic acid (AA). The AA signal has been incorporated into the background, so that the trace in Fig. 9C shows the change in voltammetric signal due to the addition of DA. The traces in Figs. 9B and C are essentially identical, showing that the presence of AA has not affected the size or shape of the DA signal. The reverse is also true; Fig. 9D shows the SD signals for
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200 FM AA in PBS alone whereas Fig. 9E shows the signal for the AA in PBS already contaiuing 1 PM DA. Again, the two signals are identical, showing that no interaction between DA and AA has occurred. It can be seen from Figs, 9D and E that the AA signal is a monophasic oxidation wave, and is much smaller on the second SD trace than the first. Ascorbic acid is hydrolysed extremely rapidly after electro-oxidation, and this makes the oxidation irreversible under voltammetric conditions. Also, it takes an appreciable time (several tens of ms) for fresh ascorbate to diffuse to the electrode surface to replace the oxidised material. It has been shown 1291 that this depletion phenomenon can be used to measure ascorbate levels by using two FCV scans, one immediately after the other. Ascorbate will appear on the first but is depleted on the second. The double scan technique in FDRV means that it can also be used for this kind of depletion analysis. After the first scan in the dual FDRV scan epoch, the solution around the surface of the carbon fibre is depleted of ascorbic acid. When the second scan occurs 10 ms later, there has not been time for diffusion to replenish the level fully. A very much smaller ascorbate signal is seen on the SD trace generated from the second and fourth scans as compared to that from the first and third scans. On the other hand, dopamine signals can be seen (Figs. 9B, C) to have equal mag~tude on both SD traces. In vitro, there is some replenishment of ascorbate and a small ascorbate signal is seen on the second SD scan. In vivo, diffusion is more restricted, and negligible replenishment occurs. This means that in vivo, the second SD trace will be essentially free of cont~nation by ascorbic acid (or other non-r~ucible electroactive materials), and will contain only signals from re-reducible materials, such as dopamine. If the two SD traces are in turn stored digitally and the second one subtracted from the first, then the difference between them will be due to the non-reducible material only. This is illustrated in Figs. 9F and G. Figure 9F shows the SD signal obtained relative to PBS (with a new electrode) after addition of both 200 PM AA and 1 FM DA. The first of the pair of signals is the sum of the faradaic signals from DA and AA; the second signal is from DA alone. Figure 9G shows the signal obtained from subtracting the two signals in Fig. 9F; it consists of an AA signal with no DA component. Thus the dual FDRV scan can differentiate DA and AA within each scan epoch. Identification of compound Pure solutions of different electroactive compounds produce different o~dation/reduction wave “‘signatures” in FDRV. It can be seen from Fig. 9 that DA and AA produce very different waveforms on the SD scans. Rapid-scan techniques are unlikely ever to be able to compete with techniques like HPLC with push-pull cannulation for detailed analysis of mixtures of substances released in viva 1301, but if one compound is present in excess, then identification from waveshape may be possible. Figure 10 shows some representative SD traces for four compounds of neurophysiological significance. Serotonin (2 X lo-’ M), for example, has a smaller and broader reduction wave than its oxidation wave; dopamine (5 x 10F7 M), in contrast, has the opposite. 5-Hydroxyindoleacetic acid (5-HIAA)
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5ms Fig. 10. Compound “signature9 in FDRV. (A) Voltage scans. (B) 2 X lo-’ DA; (D) 5 x lo-+’ M 5-HL4A; (E) 1 x 1O-5 DOPAC.
M 5-W
(C) 5 X lop7 M
(5 x lop6 M) has a broad oxidation wave but a sharp reduction wave, whereas 3,4_dihydroxyphenylacetic acid (DOPAC) (low5 M) has a clear oxidation wave but a poorly defined reduction wave. FDRV scans are considerably less sensitive to the metabo~tes of the amines than to the amines themselves; as shown in Fig. 10, the sensitivity for this electrode to DOPAC was about 5% of that to DA and the sensitivity to 5-HIAA was about 2% of that to 5-HT. It should also be noted that these waveshapes were all obtained with a scan from 0 to 900 mV positive, then down to - 300 mV negative, followed by a 200 mV negative step and a subsequent scan to 700 and -500 mV respectively. Detection or rejection of a particular compound may be optimised by different scan parameters. DISCUSSION
GeneraI features of fast-scan voltammetry methodrr in vivo Fast Cyclic Volt~et~ (FCV) has been extensively discussed elsewhere f3,4,18]. The waveforms used in FDRV are derived from those used in FCV, and the factors controlling the shape of the of the faradaic signals that are obtained are similar in both techniques. The cycle of oxidation and reduction is rapid (typically between 2 and 10 ms), and so there is little time for diffusion exchange to occur between material on the surface of the working electrode and the bulk solution. The shape of the faradaic oxidation current waveform obtained is therefore controlled by the kinetics of the oxidation reaction on the working electrode surface (‘“kinetic control”) rather than the diffusion time of new substrate to the electrode surface (“diffusion control”). The rate-of-change (rise time) of the voltage in an FDRV scan
is critical. Too fast a rise time injects too much current into the tissue and can affect the concurrently recorded nerve cells. Too slow a rise time means that oxidised material can diffuse away from the electrode before the re-reduction scan. After exhaustive testing we have found that rise times of between 1 and 3 ms (i.e. dv/dt between 1000 and 300 V/s) give the best overall performance. For many substances that are electro-o~disable at carbon fibre electrodes in aqueous solutions, the initial oxidation reaction is reversible under fast-scan conditions and a second faradaic current wave due to re-reduction of the oxidised material will be observed when the working electrode goes to a negative potential at the end of each scan. This rapid re-reduction process confers several advan~ges in vivo. Firstly, the process restores the original levels of the substrate after each analysis. Depletion of neurotrans~tter by non-reple~shi~g voltammet~c techniques could clearly have physiological consequences. Perhaps even more importantly, oxidation of certain ne~otrans~tt~rs produces compounds which are known to be toxic, for example the adrenochrome and dopachrome compounds formed from ~oradrenaline and dopamine [31]; the re-reduction cycle in rapid-scan methods prevents release of these toxic products into the environments of the electrode. The re-reduction process also prevents poisoning of the electrode surface by any insoluble products of oxidation. Use of a micro-refermce eIeclrode The micro-reference electrode monitors potentials occurring at the locus of the working electrode. These potentials are added to the signal from the macro-reference ,electrode, and this enables the system to compensate for endogenous transient signals due to local neuronal activity. Extracellular action and synaptic potentials can be recorded easily with carbon fibre electrodes 132-351, and can be shown to have amplitudes of up to several mV. These potentials are large enough to limit the resolution of the electrochemic~ analysis unless compensated for. The carbon fibre ~cro-reference could in theory be dc-coupled to the reference circuit, but because of the capacitative nature of the electrode, it has effectively infinite impedance and therefore infinite noise at dc. To overcome this problem, the micro-reference is capacitatively coupled through a simple first-order high-pass filter to the reference circuit. The macro-reference electrode has good de recording characte~st~cs, and is used to set the de reference level of the system. A total reference signal is then synthesised from the sum of the macr~reference signal and the high-pass filtered micro-reference signal. Of course, if the micro-reference electrode could be a low-impedance silver/silver chloride wire, then the macro-reference could be dispensed with and a single micro-reference used. Plotsky [36] managed to construct a m~croel~trode with two carbon fibres and a silver/silver chloride wire at the tip. The two carbon fibres served as working and auxiliary electrodes, with a single reference signal from the silver wire. We have attempted similar designs, but have found that a reference electrode made in this way is always noisy and suffers from hum and mains pick-up in our laboratory. The split-reference system is admittedly a compromise, but one which seems to work, and is relatively easy to fabricate. For
41
work in vitro or in brain slices where changes in impedance between working and auxiliary electrodes are likely to be small, the micro-reference is probably not necessary. Differential scan system The reasons for the signal subtraction procedures used in FDRV are to minimise the effect of changes in the “background” or charging current that is present in all fast voltammetric techniques. In FDRV the background current through the working electrode can be two or more orders of magnitude greater than the faradaic current produced by electroactive materials found in vivo. Thus any slight changes in background current will swamp or distort the faradaic signal current. The Helmholtz double layer at the surface of the working electrode generates an impedance which is mainly capacitative at the voltages and frequencies used in FDRV. This therefore produces a background signal which is proportional to dV/dt, the rate of change of scan voltage, rather than V itself. Because the scan voltages in the first and second halves of the FDRV epoch are identical in dV/dt, the background signal will be similar in both halves. The scans in the second half of the epoch are however generated from a baseline that is offset negative by 200 mV. Thus these scans reach the potential for faradaic oxidation later relative to the start of the scan than those in the first half. Thus the faradaic signals will be later (relative to the start of the scan) in the second half of the epoch than in the first. When the differential scans are computed, most of the background signal, which is common to both halves of the scan, is cancelled out, but the faradaic oxidation and reduction waves are converted to a biphasic (pseudo-differentiated) form.
Quadruple scan system in dual FDRV Dual mode FDRV is essentially similar to single FDRV, but with two scans in each 20 ms half-epoch instead of one. The background signal in FCV can be observed to vary as a function of the interval between scan epochs, as well as varying with pH, temperature, etc. The dependance of background signal on the interval between scan epochs means that in FCV, epoch intervals must be precisely constant: scans cannot be triggered at uneven intervals. However, with the double scan system of dual FDRV, the second scan of a pair always occurs exactly 5 ms after the end of the first, regardless of the interval between epochs. If the second of the pair of scans is used for electrochemical measurements, scan epochs may be triggered over a reasonable range of irregular intervals and a stable signal can still be obtained. Even at constant scan epoch intervals the background signal from the second SD scan is generally more stable in vivo than the signal from the first. The other advantage of dual FDRV is that it makes the separate analysis of non-re-reducible and re-reducible compounds possible, and so, for example, dopamine and ascorbate can be separately identified in a mixture of the two. It is worth noting that, even with Nafion coated carbon microelectrodes, PM levels of DA cannot be separated from 100 I_IM ascorbate using conventional FCV techniques [37]. In
48
contrast, it can be seen from Fig. 9 that with FDRV, be separately identified and measured.
2 PM DA and 200 PM AA can
Summary In the introduction to this paper it was argued that, to be applicable in vivo, a voltammetric technique must fulfill certain criteria. In essence, these were that the technique must not interfere with the normal workings of the tissue under investigation, and that the technique must be able to cope with any fluctuations in the electrical environment of the electrodes due to this normal working. Fast scan voltammetric techniques like FCV and FDRV can be shown not to interfere with neuronal activity [18], while the use of a micro-reference electrode and the differential scan method in FDRV means that this technique can also cope with the fluctuations of endogenous potential and impedance that have previously limited the resolving power of FCV in-vivo. The use of quadruple scans in dual FDRV also enables separate quantification of different electroactive materials to be made. Thus, it may be hoped that Fast Differential Ramp Voltammetry will prove a powerful method for the in-vivo analysis of synaptic dynamics and monoamine neurotransmitter function. ACKNOWLEDGEMENTS
We would like to thank T.G. Bamett for his help in constructing much of the apparatus used in these experiments. This work was supported by the Parkinson’s Disease Society. REFERENCES 1 J.B. Justice in J.B. Justice (Ed.), Voltammetry in the Neurosciences, Humana Press, Clifton, NJ, 1987, p. 3. 2 C.A. Marsden (Ed.), Measurement of Neurotransmitter Release In-Vivo, Wiley, New York, 1984. 3 J.A. Stamford, Brain. Res. Rev., 10 (1985) 119. 4 J.A. Stamford, J. Neurosci. Meth., 17 (1986) 1. 5 P.T. Kissinger, J.B. Hart and R.N. Adams, Brain Res., 55 (1973) 209. 6 R.N. Adams, Anal. Chem., 48 (1976) 1128A. 7 R.N. Adams, Trends Neurosci., (Dee 1978) 160. 8 R.N. Adams, Ann. N.Y. Acad. Sci., 473 (1986) 42. 9 J.O. Schenk and R.N. Adams in ref. 2, p. 193. 10 J.-L., Ponchon, R. Cespuglio, F. Gonon, M. Jouvet and J.F. Pujol, Anal. Chem., 51 (1979) 1483. 11 A.G. Ewing, M.A. Dayton and R.M. Wightman, Anal. Chem., 53 (1981) 1842. 12 R.F. Lane and A.T. Hubbard, Anal. Chem., 48 (1976) 1287. 13 F.G. Gonon, F. Navarre and M.J. Buda, Anal. Chem., 56 (1984) 573. 14 J.B. Ranck Jr., Brain Res., 98 (1975) 417. 15 J.B. Ranck Jr. in M.M. Patterson and R.P. Kesner (Eds.), Electrical Stimulation Research Techniques, Academic Press, New York, 1981, p. 1. 16 B. Gustafsson and E. Jankowska, J. Physiol. (London), 258 (1976) 33. 17 J.B. Ranck Jr., Exp. Neurol., 7 (1963) 153.
49 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
M. Armstrong-James and J. Millar in ref. 2, p. 209. J. Miltar and T.G. Bamett, J. Neurosci. Meth., 25 (1988) 91. M. Armstrong-James and J. Millar, J. Neurosci. Meth., 1 (1979) 279. J. Milhu and G.V. Williams, in preparation. J. Miltar, J.A. Stamford, Z.L. Kruk and R.M. Wightman, Eur. J. Pharmacol., 109 (1985) 341. A.G. Ewing, J.C. Bigelow and R.M. Wightman, Science, 221 (1983) 169. 5.4. Stamford, Z.L. Kruk and J. M&r, Brain Res., 381 (1986) 351. T. Zetterstrom, T. Sharp, C.A. Marsden and U. Ungerstedt, J. Neurochem., 41 (1983) 1769. A.W. Stemson, R. McCreery, B. Feinberg and R.N. Adams, J. Electroanal. Chem., 46 (1973) 313. M.A. Dayton, A.G. Ewing and R.M. Wightman, Anal. Chem., 52 (1980) 2392. C.R. Freed and H. Echizen, Abstr. Sot. Neurosci., 9 (1983) 999. J.A. Stamford, Z.L. Kruk and J. Millar, J. Neurosci. Meth., 10 (1984) 107. R.D. Myers, Ann. N.Y. Acad. Sci., 473 (1986) 21. P.K. Singal, K.S. Dhillon, R.E. Beamish, N. Kapur and N.S. Dhalla, Br. J. Exp. Pathol., 63 (1982) 167. M. Armstrong-James and J. Millar, J. Physiol. (London), 305 (1980) 65P. M. Armstrong-James and J. Millar, J. Physiol. (London), 308 (1980) 116P. J. Millar and M. Armstrong-James, Brain Res. 231 (1982) 267. J. Millar and M. Armstrong-James, Brain Res., 243 (1982) 253. P.M. Plotsky, Brain Res., 235 (1982) 179. D.J. Wiedemann, A. Basse-Tomusk, R.M. Wightman and G.V. Rebec, Sot. Neurosci. Abstr., 14 (1988) 294.
J. Electroanal. Chem., 282 (1990) 33-49 Elsevier Sequoia S.A., Lausarme - Printed
in The Netherlands
Fast differential ramp voltammetry: a new voltammetric technique designed specifically for use in neuronal tissue J. Millar and G.V. Williams Dept. of Physiology, (Received
Turner Street, London El 2AD (Great Britain)
13 July 1989; in revised form 13 November
1989)
ABSTRACT This paper describes details of a new voltammetric technique designed specifically for use in living, and the function of the especially neuronal, tissue. Neuronal tissue is an “active” electrical environment, nerve cells may be disrupted by some voltammetric procedures. The new technique, which is called “Fast Differential Ramp Voltammetry (FDRV)“, has been developed from Fast Cyclic Voltammetry (FCV). FCV can be shown not to interfere with ongoing neuronal activity because the working electrode can be switched to record the electrical activity of the surrounding nerve cells in between voltammetric scans. The charging currents generated in FCV are however affected by local neuronal activity, and in the past this has limited the resolution of FCV in viva. In FDRV these effects are reduced by a double or quadruple offset scan technique which cancels out most of the variation in signal due to fluctuations in charging currents. The resolution of FDRV should be adequate to study “basal” levels of neurotransmitters in viva. The technique is immune to electrocatalytic effects and can resolve separate peaks for mixtures of re-reducible and non re-reducible electroactive compounds. This enables it to be used for separate measurement of, for example, dopamine and ascorbic acid in mixtures of the two.
INTRODUCTION
The last decade has seen the development of voltammetry at carbon fibre electrodes into an important tool for the investigation of neurotransmitter function in the mammalian brain (see refs. l-4 for reviews). This development has largely stemmed from the work of Ralph Adams and his colleagues in the early 1970’s, who first applied voltammetric methods to the study of neurotransmitter release in vivo [5-S]. A fundamental characteristic of voltammetry is that the analysis requires the application of a voltage waveform to the tissue being studied. In particular, many voltammetric techniques use voltage pulses as part of the analytical waveform. These include chronoamperometry [9] normal pulse voltammetry [lO,ll] differential pulse voltammetry [12], and differential double pulse voltammetry [12,13]. However, 0022-0728/90/$03.50
0 1990 Elsevier Sequoia
S.A.
34
voltage pulses are normally used by neurobiologists as a means of st~ulating nerve cells, and it is known that currents of less than 1 pA can activate a cell under the right conditions [14-161. Thus, before most neurobiologists will accept the validity of in-vivo voltammetric data, they will need to be convinced that the currents flowing through the working and auxiliary electrodes do not affect the normal functio~ng of the tissue. In other words, the applied waveforms must be shown unequivocally not to stimulate or block the activity of nerve cells. The cells must be recorded as close to the working electrode as possible, for that is where the current density will be greatest; it is not adequate to show that cells at a distance from the working electrode are unaffected, although if distant cells were affected, obviously the technique would be unacceptable. A second major consideration for in-vivo volt~et~ is the fact that the brain is an “active” electrical environment, in the sense that the neurones and glial cells produce ac and dc voltages as part of their normal function. There may also be spontaneous changes in tissue impedance due to the activity of the cells [17]. Thus a second requirement for a voltammetric technique to be used in vivo is that it must allow for and accommodate the normal fluctuations in potential and impedance found in living tissue. The third and final consideration will involve the size of the electrodes that are used. The diameters of the cell bodies of nerve cells lie mostly in the range of 10 to 50 pm. These cells are packed closely together with their supporting glial cells such that the extracellular space in neuronal tissue is normally no more than about lo-20% Any electrode inserted into this tissue will cause damage, but if the electrode diameter can be made comparable to or smaller than the typical cell diameter, then it can be hoped that the damage will be confined to the cells directly in the path of the electrode. Larger electrodes will not only destroy the cells directly in their path, but will also inflict “crush injuries” on cells lateral to the electrode. In this paper we describe fast differential ramp voltammetry (FDRV), using 8 pm diameter carbon fibre microelectrodes. This technique has been developed from fast cyclic voltammetry (FCV) 118,191,specifically as an attempt to satisfy the conditions set down above. A special feature of FDRV is the elimination of electrocatalytic effects in the analysis without alteration of normal brain levels of electroactive materials, and thus the possibility of simultaneous independent quantification of catecholamine and ascorbic acid in mixtures of the two. EXPERIMENTAL
Electrodes and potentiostat
configuration
A four-electrode system is used, comprising a working, auxiliary, and two reference electrodes. The two reference electrodes are designated micro-reference and macro-reference. The working and micro-reference electrodes are made from carbon fibres, incorporated together into a compound glass microelectrode. The construction of this compound carbon fibre electrode is similar to that previously described for single electrodes [20], except that the glass blank is a theta or “quad”
35
I
I 20 pm
Fig. 1. Appearance of the tip region of a twin carbon fibre microelectrode. The glass insulation is on the left, with the exposed fibres, cut or spark etched to the appropriate length, protruding on the right. (A) Working electrode; (B) micro-reference electrode.
type. (Theta glass tubing has a septum down the middle so that in cross-section it looks like the Greek letter theta; two microelectrode “barrels” can thus be obtained from one piece of glass when it is pulled into a microelectrode. Quad or quadrant glass contains a septum that that is cruciform in cross-section and thus divides the internal space of the tube into four sections, like a pie chart. This can be pulled to make a four-barrel electrode.) Single carbon fibres are inserted into two compartments in the tube, which can then be pulled in a microelectrode puller to leave the two fibres protruding from two separate barrels beyond the glass at the tip. The fibres are either cut mechanically or etched with a high-voltage spark [21] to a tip length of 20 to 50 pm, but receive no other “pre-treatment” or coating. Contact with the fibres in the blunt end of the electrode can be made with a fine copper wire dipped in silver-doped epoxy glue. Figure 1 shows the appearance of the compound electrode tip in a two-barrel electrode. The macro-reference electrode is a conventional silver/ silver chloride electrode and the auxiliary electrode (which may in theory be of any conducting material) is in practice usually either another Ag/AgCl electrode or a stainless steel wire. The micro-reference is capacitatively coupled through a first-order high-pass filter to the reference circuit. The macro-reference electrode should be placed on the surface of the brain, or at some other point where high frequency changes in potential do not occur. A total reference signal is then synthesised from the sum of the macro-reference signal and the high-pass filtered micro-reference signal. In order to obtain extracellular voltage recordings in the intervals between voltammetric scan epochs, and to enable multiple working electrodes to be used asynchronously in the same preparation, the usual voltammetric system of an active auxiliary electrode and a passive working electrode is reversed in FDRV. The auxiliary electrode is permanently connected to ground (earth) potential, while the driving voltage waveform is applied to the working electrode. We call this the “grounded auxiliary system”. The total potentiostat using this grounded auxiliary system and the compound reference circuit is shown in Fig. 2. Voltammetric scan parameters; the “time share” recording system This is identical in principle to that used for FCV. The working electrode is connected to the voltammetric amplifier for only 40 ms at a time. In between these
I
Logic Drive to Analog Gate
Voltammetry
1 Micro-reference
OUtPUt
Electrode
Jp!G
Macro-reference
160K
47 M
J..> i
\
Preparatlon
u
-L
IOK -
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Fig. 2. The four-electrode potentiostat circuit for Fast Differential Ramp Voltammetry (FDRV). Analog gate: l/2 DG303 or similar. Operational amplifiers: (1, 2, 5, 7) OPAlll; (3, 4, 6, 8) 0PA2111 or similar. Resistors: 0.1% metal film (1% for 1 and 47 I@.
40 ms “scan epochs” the working electrode is switched electronically to the input of a voltage-follower amplifier. The voltage signal obtained from this follower can then be amplified and filtered to give a record of the ongoing electrical activity of the nerve cells in the intervals between voltammetric scan epochs [18]. By recording the electrical activity of the nerve cells immediately before and after each voltammetric scan epoch it can be seen whether or not the electrochemical analysis has affected the spontaneous activity of the neurones. In our present studies, the scan epochs are repeated at intervals of 250 or 500 ms. Thus at 500 ms intervals, one obtain 450 ms periods of voltage recording (500 ms between epochs minus 10 ms “settling time”) alternating with 40 ms of voltammetric recordings. Fast Differential Ramp Voltammetry (FDR V) FDRV uses fast triangular waves (“ramps”) to oxidise and reduce the electroactive material in solution. These waves are biphasic, positive-negative going waveforms. FCV uses a triphasic waveform, but the initial negative-going part of the FCV wave does not seem to be necessary and may be discarded. A single biphasic wave can, for the sake of clarity, be referred to as a “scan”. FDRV can be carried out in two ways: single or dual mode. In single FDRV a pair of scans is generated in each scan epoch (Fig. 3A). The scan in the second 20 ms is an exact repeat of the scan in the first 20 ms, but offset negative by a fixed voltage step, usually 200 mV. The amplitude of the positive and negative peaks within the scan are variable (by an
37
Fig. 3. Drive waveforms and background signals in FDRV. (A) Twin voltage ramp patterns (scans) used in single model FDRV. (B) Background current generated by these waveforms when a carbon fibre working electrode is immersed in phosphate-buffers saline (PBS). (C) The two waveforms in (B) electronically subtracted to give a “~fferenti~ signal. (D) A ~fferenti~ signal with a earher “standard” differential signal subtracted from it; this is the “subtracted differential” or SD signal. (E) SD signal from (D) at x 50 gain. Calibration bar: (A) 400 mV, (B, C, D) 100 nA, (E) 2 nA.
analog amplifier) but the duration is always 10 ms. Thus the rate of change of voltage, dV/db, varies as a function of the ~p~t~de. For an ~p~tude of f 1 V, dV/dt would be 400 V/s. In practice, a positive peak of f 900 mV, and a negative peak of - 300 mV has been found suitable for detection of most catecholamines (in later experiments, some improvement in detection in vivo was found with scans to -t 1.4 V instead of 900 mv). Figure 3A shows a pair of voltage scans generated in one scan epoch. The corresponding background currents generated in the carbon fibre working electrode by these voltages are shown underneath, in Fig. 3B. The working electrodes were immersed in phosphate-buffered 0.9% saline (PBS) at room temperature. These background currents are due to the electrical properties of the double layer at the electrode/electrolyte interface, and are extremely stable at a fixed temperature and pH. The current waveform through the working electrode is converted to a voltage by an operational amplifier used as a current-to-voltage converter (1 in Fig. 2) then amplified to a s&able level (approx. &5 V), digit&d with a precision 12-bit ~~og-to-~~t~ converter and stored el~tro~c~ly. The signal produced during the scan in the second half of the scan epoch is then subtracted digitally point-by-point from the signal produced during the first scan to give a “differential” signal (Fig. 3C). As can be seen, this procedure cancels out most of the backwood signal. The differential signal is then in turn stored and forms a standard or “reference” differential signal. The standard differential signal is subtracted digitally from the differential signal generated in the following scan epoch to give a “subtracted differential” (SD) signal. If no changes have occurred in the solution between the acquisition of the standard and present differential signals, then the SD signal is a
Fig. 4. Background signals from an FDRV scan with and without micro-reference compensation. (A) Drive waveform. (B) Signals in PBS. (C) Signals in rat caudate nucleus. (D) Signals in rat corpus callosum.
straight line (Fig. 3D). This two-stage subtraction process eliminates the background current, but allows faradaic signals to show through, although in an unusual form (see below). To illustrate the noise level of the system, Fig. 3E shows the SD signal at X 50 gain. The noise current was found to depend on tip size; most electrodes generated a noise (peak to peak, 0 to 5 KHz) of between 100 and 1000 pA. The importance of the micro-reference in-vivo electrode can be illustrated by its effect on the background signal. Figure 4 shows the background signals from a typical dual carbon fibre electrode as illustrated in Fig. 1. The voltage scan (Fig. 4A) was + 900 to - 300 mV in 5 ms. Figure 4B shows two superimposed traces obtained in PBS, one with and one without the micro-reference connected. The signal with the micro-reference in circuit is about 5% greater, implying that the impedance of the electrolyte between the working and auxiliary electrodes is a significant fraction of the total impedance. Figure 4C shows the backgrounds from the same electrode with and without the micro-reference with the electrode in the caudate nucleus of an anaesthetised rat; the difference is similar to that seen in vitro. Figure 4D shows the signal with the electrode in the corpus callosum. In this region, the high density of myelinated axons in the neuropil increases the tissue impedance and the signal is considerably attenuated without the micro-reference in circuit. Even in regions
where the impedance is low, the micro-reference is able to compensate for changes in impedance due to neuronal activity, and thus stabilise the charging current signal. Addition of electroactive materials to the solution around the working electrode will generate faradaic oxidation and reduction signals. This is illus~ated in Fig. 5. Figure 5A shows a pair of scan voltages as in Fig. 3A. Figure 5B shows two superimposed current traces obtained first in PBS and then with 20 pm dopamine (DA) present in the PBS. An increase in signal current is visible at two regions; firstly at a positive-going (outsing) voltage and then at a negative-gong (reducing)
39
Fig. 5. Faradaic currents from FDRV scans. (A) Drive waveforms. (B) Electrode current before and after addition of dopamine (DA} to give 20 pM final concentration. (C) The two differential signals obtained from the traces in (B). (D) A standard differential signal from the difference between the two signals in (C). (E) SD signal in {Dj at ten times gain. Calibration bar: (A) 400 mV, (B, C, D) 100 nA, (E) 10 ILL.
one. Figure 5C shows the appearance of the differential signal before and after addition of dopamine and Fig. SD shows the corresponding SD signal (i.e. the difference between the two signals in Fig. 32). Finally, Fig. 5E shows the SD signal at X 10 gain. In Figs. 5D and E both the faradaic oxidation wave and the reduction wave have a biphasic, “pseudo-differentiated” appearance. The reason for the biphasic appearance of the faradaic oxidation and reduction waves in the SD signal is that the the signal in the second half of the epoch is generated from a negative-offset form of the drive waveform; since this waveform is a positive-going ramp, it reaches the oxidation potential for dopamine later (relative to the start of the ramp) than in the first scan. In Fig. 53, the dopamine oxidation signal can be seen to be displaced on the background in the second scan as compared to the first scan. When the signals in the first and second halves of the epoch are subtracted, the backgrounds are s~chro~sed and tend to cancel out but the faradaic signals are time-shifted and so the monophasic oxidation and reduction waves are converted to a biphasic, ‘“pseudo-differentiated” form.
Single and dual mode FDRV differ only in the pattern and timecourse of the voltammetric scans used in the 40 ms scan epoch. In dual FDRV, four biphasic scans with faster rise times are used, each one lasting 5 ms, with the last pair offset negative from the first pair. Thus, two subtracted differential signals are obtained
Fig. 6. Dual mode FDRV. (A, B) Voltage and current waveforms similar to Fig. 4, but with two scans at each baseline voltage. (C, D) Differential and standard differential signals for 50 pM DA in PBS. (E) SD signal from (D) at X 10 gain. Calibration: (A) 400 mV, (B, C, D) 200 nA, (E) 20 nA.
per scan epoch, as shown in Fig. 6 (Figs. 5 and 6 both show the signal from 20 pm DA with the same scan amplitudes and offset). In mixtures of electroactive materials or in vivo, dual FDRV has several advantages over single FDRV, which are discussed below. Data collection and analysis
The SD signal is generated once (single mode) or twice (dual mode) per scan epoch in FDRV, at scan epoch rates of up to 16 SK’. This signal may be stored on tape if a wide-bandwidth tape recorder is available, but in fact much of the information in the trace is redundant if a only single electroactive species is changing in concentration at any one time. In this case a reduced data collection system may be used, where the amplitude of the faradaic oxidation or reduction wave is sampled at a fixed voltage on a single scan in each epoch, and this voltage is held between scans by means of a sample-and-hold circuit. This form of data sampling has been used extensively in studies using FCV [22]. A considerable improvement in signal-to-noise ratio is possible in FDRV if two S&H circuits are used to sample the peak and trough of the (biphasic) oxidation wave. The signals from the two circuits are fed into a difference amplifier to give the peak-to-peak amplitude of the SD oxidation wave. This procedure nearly doubles the absolute sensitivity of the sample, and, more importantly, cancels out any low frequency drift
41
Fig. 7. Comparison of backwood-subtracts FCV (A) and subtracted differential FDRV (B) signals for 50 nM dopamine in vitro. Dots mark where signals are sampled by sample-and-bold circuits to provide continuous outputs to a chart recorder. (C) Drive waveform for both (A) and (B) signals.
in the sampled signal. Figure 7 shows a comparison of two 50 nM dopamine signals. Figure 7A shows an FCV scan with the background subtracted to show the faradaic oxidation and reduction waves generated by the scan voltage in Fig. 7C. This signal rides on a backwood noise level which can be estimated by subtracting the backgronnd from itself; this is shown as the nearly flat line in Fig. 7A. A single S&H circuit would be most sensitive if sampling at the point shown by the dot, but low fr~uency drift means that the signal-to-noise ratio is only about two to one_ Figure 7B shows the same concentration analysed with FDRV and using a dual S&H circuit at the points shown. Baseline drift is now minimal and the signal to noise ratio is now about five to one. RESULTS
In st~d~d ~o~~ti~ns in vitro the sensitivity of FDRV for most electrodes was found to lie in the region of 10 nM for dopamine. This was the concentration that would produce a signal at the oxidation peak current of approximately double the noise level. Occasionally electrodes were made that could detect less than 5 nM dopamine; these electrodes had very low noise levels; the reason for this has not yet been established. “Standard conditions” can be defined as PBS which was continually stirred by air bubbled through it, m~ntained at a fixed temperature between 15 and 20” C, with the test electrode having stabilised its background by continuous scans at 2 Hz or faster for at least 10 mm before testing. All tests were done with scan epochs repeated at 2 Hz. No ascorbate or other reducing agents were added to the solution. Figure 8A shows the voltage scan for the fourth of a dual FDRV set. Figure 8B shows the SD signal produced on this scan by the addition of 10 nM dopamine to the test solution. Six trials were carried out on a single electrode, washing out the beaker and electrode between trials and allowing the background to stabilise for 5 min before addition of dopamine. The signals 10 s after addition of
42
Fig. 8. In-vitro FDRV detection thresholds. (A) Drive waveform. (B) SD signals for 10 nM dopamine in PBS; six traces supe~m~sed. (C) Dual S&H records for a series of additions of 10 nM increments of dopamine to PBS (each addition produces a signal similar to that shown in (B)). (D) Similar data to (C) with a different electrode. Time calibration (A, B) 1 ms, (C, D) 2 min.
dopamine (to allow for mixing) are shown superimposed. A dual sample-and-hold circuit was used to capture the amplitude of the signals at the times marked by dots. Figures 8C and D show the dual S&H signals obtained from two further electrodes. Dopamine was added at the points marked by dots to give a series of IO nM increments in concentration. Each increment of dop~ne can be seen to produce a clearly visible increase in the sampled signal level. In vivo, the ongoing electrical and chemical activity of the nerve cells would be expected to increase the background noise and thus decrease the detectabi~ty of amines. However, when investigating dopamine release in the rat striatum following electrical stimulation of the nigro-striatal pathway [22-241, we have detected changes of less than 20 nM dopamine [21]. Thus FDRV appears to have sufficient sensitivity to detect basal levels of catecholamines in the mammalian brain, which appear to be of the order of 50 nM [25]. Selectivity One of the major problems with many voltammetric techniques is the difficulty of identifying the individual components in a mixture of electroactive materials. In particular, the phenomenon of “electrocatalysis” [26-281 can cause non-linear summation or multiplication of oxidation signals in mixtures of catecholamines and ascorbic acid. This happens when a molecule of catecholamine is re-reduced by a molecule of ascorbate immediately after electro-oxidation at the working electrode. The re-reduced catecholamine may then be oxidised again on the electrode, and a cycle of reactions set up, with the net result that the ascorbate effectively amplifies the dopamine signal, or at least produces a non-linear addition of ascorbate and
43
Fig. 9. Absence of electrocatalytic effects in dual FDRV. (A) Dual FDRV voltage scans. (B) SD signals for 1 pM DA in PBS. (C) Similar signals for 1 pM DA in PBS containing 200 pM ascorbic acid (AA). (0) SD signal for 200 pM AA in PBS. (E) SD signal for 200 pM AA in PBS containing 1 pM DA. (F) SD signals (with a new electrode) for a mixture of 200 FM AA and 1 pM DA relative to PBS. (G) Result of subtraction of the second SD signal in (F) from the first.
dopamine signals. Electrocatalytic effects are significant in slow-scan cyclic voltammetry, but get smaller with faster scan rates, because there is a shorter total time allowed for oxidation. In normal FCV electrocatalytic effects have been found to be small, and the faradaic signal produced by a mixture of ascorbate and dopamine is approximately the sum of the individual signals generated by separate solutions (unpublished results). The higher speed of the scans in FDRV as compared to FCV is sufficient to eliminate electrocatalytic effects completely. It has been calculated [27] that the quinone initial product of dopamine electro-oxidation has a half-life of 2.1 ms in the presence of ascorbate. To obtain electrocatalysis, the quinone must be reduced and then the dopamine re-oxidised. Inspection of dual FDRV traces shows that the dopamine oxidation takes about 2 ms. Thus a reasonable minimum time for a cycle of electrocatalysis to occur would be about 6 ms. However, since the total time for oxidation in dual FDRV is less than 4 ms, there is insufficient time for electrocatalysis to occur, and dopamine and ascorbate signals sum linearly. Figure 9 illustrates this effect. Figure 9A shows the last pair of voltage scans in an FDRV set; Fig. 9B shows the SD signals for 1 PM DA in PBS. Figure 9C shows similar data for the same electrode with the same concentration of DA but this time added to PBS already containing 200 PM ascorbic acid (AA). The AA signal has been incorporated into the background, so that the trace in Fig. 9C shows the change in voltammetric signal due to the addition of DA. The traces in Figs. 9B and C are essentially identical, showing that the presence of AA has not affected the size or shape of the DA signal. The reverse is also true; Fig. 9D shows the SD signals for
44
200 FM AA in PBS alone whereas Fig. 9E shows the signal for the AA in PBS already contaiuing 1 PM DA. Again, the two signals are identical, showing that no interaction between DA and AA has occurred. It can be seen from Figs, 9D and E that the AA signal is a monophasic oxidation wave, and is much smaller on the second SD trace than the first. Ascorbic acid is hydrolysed extremely rapidly after electro-oxidation, and this makes the oxidation irreversible under voltammetric conditions. Also, it takes an appreciable time (several tens of ms) for fresh ascorbate to diffuse to the electrode surface to replace the oxidised material. It has been shown 1291 that this depletion phenomenon can be used to measure ascorbate levels by using two FCV scans, one immediately after the other. Ascorbate will appear on the first but is depleted on the second. The double scan technique in FDRV means that it can also be used for this kind of depletion analysis. After the first scan in the dual FDRV scan epoch, the solution around the surface of the carbon fibre is depleted of ascorbic acid. When the second scan occurs 10 ms later, there has not been time for diffusion to replenish the level fully. A very much smaller ascorbate signal is seen on the SD trace generated from the second and fourth scans as compared to that from the first and third scans. On the other hand, dopamine signals can be seen (Figs. 9B, C) to have equal mag~tude on both SD traces. In vitro, there is some replenishment of ascorbate and a small ascorbate signal is seen on the second SD scan. In vivo, diffusion is more restricted, and negligible replenishment occurs. This means that in vivo, the second SD trace will be essentially free of cont~nation by ascorbic acid (or other non-r~ucible electroactive materials), and will contain only signals from re-reducible materials, such as dopamine. If the two SD traces are in turn stored digitally and the second one subtracted from the first, then the difference between them will be due to the non-reducible material only. This is illustrated in Figs. 9F and G. Figure 9F shows the SD signal obtained relative to PBS (with a new electrode) after addition of both 200 PM AA and 1 FM DA. The first of the pair of signals is the sum of the faradaic signals from DA and AA; the second signal is from DA alone. Figure 9G shows the signal obtained from subtracting the two signals in Fig. 9F; it consists of an AA signal with no DA component. Thus the dual FDRV scan can differentiate DA and AA within each scan epoch. Identification of compound Pure solutions of different electroactive compounds produce different o~dation/reduction wave “‘signatures” in FDRV. It can be seen from Fig. 9 that DA and AA produce very different waveforms on the SD scans. Rapid-scan techniques are unlikely ever to be able to compete with techniques like HPLC with push-pull cannulation for detailed analysis of mixtures of substances released in viva 1301, but if one compound is present in excess, then identification from waveshape may be possible. Figure 10 shows some representative SD traces for four compounds of neurophysiological significance. Serotonin (2 X lo-’ M), for example, has a smaller and broader reduction wave than its oxidation wave; dopamine (5 x 10F7 M), in contrast, has the opposite. 5-Hydroxyindoleacetic acid (5-HIAA)
45
5ms Fig. 10. Compound “signature9 in FDRV. (A) Voltage scans. (B) 2 X lo-’ DA; (D) 5 x lo-+’ M 5-HL4A; (E) 1 x 1O-5 DOPAC.
M 5-W
(C) 5 X lop7 M
(5 x lop6 M) has a broad oxidation wave but a sharp reduction wave, whereas 3,4_dihydroxyphenylacetic acid (DOPAC) (low5 M) has a clear oxidation wave but a poorly defined reduction wave. FDRV scans are considerably less sensitive to the metabo~tes of the amines than to the amines themselves; as shown in Fig. 10, the sensitivity for this electrode to DOPAC was about 5% of that to DA and the sensitivity to 5-HIAA was about 2% of that to 5-HT. It should also be noted that these waveshapes were all obtained with a scan from 0 to 900 mV positive, then down to - 300 mV negative, followed by a 200 mV negative step and a subsequent scan to 700 and -500 mV respectively. Detection or rejection of a particular compound may be optimised by different scan parameters. DISCUSSION
GeneraI features of fast-scan voltammetry methodrr in vivo Fast Cyclic Volt~et~ (FCV) has been extensively discussed elsewhere f3,4,18]. The waveforms used in FDRV are derived from those used in FCV, and the factors controlling the shape of the of the faradaic signals that are obtained are similar in both techniques. The cycle of oxidation and reduction is rapid (typically between 2 and 10 ms), and so there is little time for diffusion exchange to occur between material on the surface of the working electrode and the bulk solution. The shape of the faradaic oxidation current waveform obtained is therefore controlled by the kinetics of the oxidation reaction on the working electrode surface (‘“kinetic control”) rather than the diffusion time of new substrate to the electrode surface (“diffusion control”). The rate-of-change (rise time) of the voltage in an FDRV scan
is critical. Too fast a rise time injects too much current into the tissue and can affect the concurrently recorded nerve cells. Too slow a rise time means that oxidised material can diffuse away from the electrode before the re-reduction scan. After exhaustive testing we have found that rise times of between 1 and 3 ms (i.e. dv/dt between 1000 and 300 V/s) give the best overall performance. For many substances that are electro-o~disable at carbon fibre electrodes in aqueous solutions, the initial oxidation reaction is reversible under fast-scan conditions and a second faradaic current wave due to re-reduction of the oxidised material will be observed when the working electrode goes to a negative potential at the end of each scan. This rapid re-reduction process confers several advan~ges in vivo. Firstly, the process restores the original levels of the substrate after each analysis. Depletion of neurotrans~tter by non-reple~shi~g voltammet~c techniques could clearly have physiological consequences. Perhaps even more importantly, oxidation of certain ne~otrans~tt~rs produces compounds which are known to be toxic, for example the adrenochrome and dopachrome compounds formed from ~oradrenaline and dopamine [31]; the re-reduction cycle in rapid-scan methods prevents release of these toxic products into the environments of the electrode. The re-reduction process also prevents poisoning of the electrode surface by any insoluble products of oxidation. Use of a micro-refermce eIeclrode The micro-reference electrode monitors potentials occurring at the locus of the working electrode. These potentials are added to the signal from the macro-reference ,electrode, and this enables the system to compensate for endogenous transient signals due to local neuronal activity. Extracellular action and synaptic potentials can be recorded easily with carbon fibre electrodes 132-351, and can be shown to have amplitudes of up to several mV. These potentials are large enough to limit the resolution of the electrochemic~ analysis unless compensated for. The carbon fibre ~cro-reference could in theory be dc-coupled to the reference circuit, but because of the capacitative nature of the electrode, it has effectively infinite impedance and therefore infinite noise at dc. To overcome this problem, the micro-reference is capacitatively coupled through a simple first-order high-pass filter to the reference circuit. The macro-reference electrode has good de recording characte~st~cs, and is used to set the de reference level of the system. A total reference signal is then synthesised from the sum of the macr~reference signal and the high-pass filtered micro-reference signal. Of course, if the micro-reference electrode could be a low-impedance silver/silver chloride wire, then the macro-reference could be dispensed with and a single micro-reference used. Plotsky [36] managed to construct a m~croel~trode with two carbon fibres and a silver/silver chloride wire at the tip. The two carbon fibres served as working and auxiliary electrodes, with a single reference signal from the silver wire. We have attempted similar designs, but have found that a reference electrode made in this way is always noisy and suffers from hum and mains pick-up in our laboratory. The split-reference system is admittedly a compromise, but one which seems to work, and is relatively easy to fabricate. For
41
work in vitro or in brain slices where changes in impedance between working and auxiliary electrodes are likely to be small, the micro-reference is probably not necessary. Differential scan system The reasons for the signal subtraction procedures used in FDRV are to minimise the effect of changes in the “background” or charging current that is present in all fast voltammetric techniques. In FDRV the background current through the working electrode can be two or more orders of magnitude greater than the faradaic current produced by electroactive materials found in vivo. Thus any slight changes in background current will swamp or distort the faradaic signal current. The Helmholtz double layer at the surface of the working electrode generates an impedance which is mainly capacitative at the voltages and frequencies used in FDRV. This therefore produces a background signal which is proportional to dV/dt, the rate of change of scan voltage, rather than V itself. Because the scan voltages in the first and second halves of the FDRV epoch are identical in dV/dt, the background signal will be similar in both halves. The scans in the second half of the epoch are however generated from a baseline that is offset negative by 200 mV. Thus these scans reach the potential for faradaic oxidation later relative to the start of the scan than those in the first half. Thus the faradaic signals will be later (relative to the start of the scan) in the second half of the epoch than in the first. When the differential scans are computed, most of the background signal, which is common to both halves of the scan, is cancelled out, but the faradaic oxidation and reduction waves are converted to a biphasic (pseudo-differentiated) form.
Quadruple scan system in dual FDRV Dual mode FDRV is essentially similar to single FDRV, but with two scans in each 20 ms half-epoch instead of one. The background signal in FCV can be observed to vary as a function of the interval between scan epochs, as well as varying with pH, temperature, etc. The dependance of background signal on the interval between scan epochs means that in FCV, epoch intervals must be precisely constant: scans cannot be triggered at uneven intervals. However, with the double scan system of dual FDRV, the second scan of a pair always occurs exactly 5 ms after the end of the first, regardless of the interval between epochs. If the second of the pair of scans is used for electrochemical measurements, scan epochs may be triggered over a reasonable range of irregular intervals and a stable signal can still be obtained. Even at constant scan epoch intervals the background signal from the second SD scan is generally more stable in vivo than the signal from the first. The other advantage of dual FDRV is that it makes the separate analysis of non-re-reducible and re-reducible compounds possible, and so, for example, dopamine and ascorbate can be separately identified in a mixture of the two. It is worth noting that, even with Nafion coated carbon microelectrodes, PM levels of DA cannot be separated from 100 I_IM ascorbate using conventional FCV techniques [37]. In
48
contrast, it can be seen from Fig. 9 that with FDRV, be separately identified and measured.
2 PM DA and 200 PM AA can
Summary In the introduction to this paper it was argued that, to be applicable in vivo, a voltammetric technique must fulfill certain criteria. In essence, these were that the technique must not interfere with the normal workings of the tissue under investigation, and that the technique must be able to cope with any fluctuations in the electrical environment of the electrodes due to this normal working. Fast scan voltammetric techniques like FCV and FDRV can be shown not to interfere with neuronal activity [18], while the use of a micro-reference electrode and the differential scan method in FDRV means that this technique can also cope with the fluctuations of endogenous potential and impedance that have previously limited the resolving power of FCV in-vivo. The use of quadruple scans in dual FDRV also enables separate quantification of different electroactive materials to be made. Thus, it may be hoped that Fast Differential Ramp Voltammetry will prove a powerful method for the in-vivo analysis of synaptic dynamics and monoamine neurotransmitter function. ACKNOWLEDGEMENTS
We would like to thank T.G. Bamett for his help in constructing much of the apparatus used in these experiments. This work was supported by the Parkinson’s Disease Society. REFERENCES 1 J.B. Justice in J.B. Justice (Ed.), Voltammetry in the Neurosciences, Humana Press, Clifton, NJ, 1987, p. 3. 2 C.A. Marsden (Ed.), Measurement of Neurotransmitter Release In-Vivo, Wiley, New York, 1984. 3 J.A. Stamford, Brain. Res. Rev., 10 (1985) 119. 4 J.A. Stamford, J. Neurosci. Meth., 17 (1986) 1. 5 P.T. Kissinger, J.B. Hart and R.N. Adams, Brain Res., 55 (1973) 209. 6 R.N. Adams, Anal. Chem., 48 (1976) 1128A. 7 R.N. Adams, Trends Neurosci., (Dee 1978) 160. 8 R.N. Adams, Ann. N.Y. Acad. Sci., 473 (1986) 42. 9 J.O. Schenk and R.N. Adams in ref. 2, p. 193. 10 J.-L., Ponchon, R. Cespuglio, F. Gonon, M. Jouvet and J.F. Pujol, Anal. Chem., 51 (1979) 1483. 11 A.G. Ewing, M.A. Dayton and R.M. Wightman, Anal. Chem., 53 (1981) 1842. 12 R.F. Lane and A.T. Hubbard, Anal. Chem., 48 (1976) 1287. 13 F.G. Gonon, F. Navarre and M.J. Buda, Anal. Chem., 56 (1984) 573. 14 J.B. Ranck Jr., Brain Res., 98 (1975) 417. 15 J.B. Ranck Jr. in M.M. Patterson and R.P. Kesner (Eds.), Electrical Stimulation Research Techniques, Academic Press, New York, 1981, p. 1. 16 B. Gustafsson and E. Jankowska, J. Physiol. (London), 258 (1976) 33. 17 J.B. Ranck Jr., Exp. Neurol., 7 (1963) 153.
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