Journal of Neuroscience Methods, 32 (1990) 37-44 Elsevier
37
NSM 01051
Application of fast cyclic voltammetry to measurement of electrically evoked dopamine overflow from brain slices in vitro D.R. Bull 1, p. Palij 3, M.J. Sheehan 1, j. Millar 2, J.A. Stamford 3, Z.L. Kruk 3 and P.P.A. H u m p h r e y 1 1 Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts (U. K.L and Departments of 2 Physiology and 3 Pharmacology, The London Hospital Medical College, London El 2AD (U.K.) (Received 7 August 1989) (Revised version received 5 December 1989) (Accepted 7 December 1989)
Key words: Fast cyclic voltammetry; Dopamine; Release; Striatum; In vitro Fast cyclic voltammetry at a carbon fibre microelectrode was used to monitor the time course of dopamine overflow in slices of rat corpus striatum incubated in a brain slice chamber. Dopamine release occurred in response to electrical stimulation. Electrochemical, physiological and pharmacological evidence indicates that release of endogenous dopamine can be measured reliably for up to 9 h and that fast cyclic voltammetry can be used in brain slices for quantitative studies of dopamine release in the CNS.
Introduction
Fast cyclic voltammetry (FCV) is an electrochemical detection method which has been applied to quantification of ionophoresis (Armstrong-James et al. 1980, 1981; Kruk et al. 1980; Millar et al. 1981) and to monitoring of changes of the extracellular concentration of dopamine in the central nervous system of the anaesthetised rat (Millar et al. 1985; Stamford et al. 1986a,b) and measurement of ascorbic acid distribution in the brain (Stamford et al. 1984a,b). The advantages of FCV over other voltammetric methods have been reviewed (Armstrong-James et al. 1981; Kruk 1986) and they can be summarised as measuring evoked endogenous dopamine without the addition of MAOI (as opposed to measuring metabolites), having a time resolution of better than 50 ms, and allowing pharmacological mani-
Correspondence: Dr. D.R. Bull, Dept. of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 ODP, U.K.
pulations which allow investigation of storage and release of dopamine (Kruk and Stamford 1985; Stamford et al. 1986c), high and low affinity dopamine uptake (Stamford et al. 1984a,b, 1986d) and control of release by autoreceptors (Stamford et al. 1986d,e, 1987). FCV in vivo is not without some limitations; experiments have to be done in anaesthetised animals, the sensitivity of the method does not allow measurement of basal (background) concentrations of dopamine, the range of drugs and experimental manipulation is limited to agents which can readily cross the blood-brain barrier, and generally, it is only possible to study a single dose of a drug in each experiment. As with all stereotaxic methods, the anatomical location of electrodes cannot be known accurately except by post-experimental histological examination. To overcome some of these limitations, and to extend the range of investigations possible using FCV, we have investigated whether FCV could be used in brain slices. The use of brain slices for qualitative and quantitative neurobiology is established, and the
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
38
advantages of brain slices have been extensively reviewed (Kerkut and Wheal 1981; Dingledine 1984). In applying FCV in brain slices, the ability to control accurately the concentration of drug to which the tissue is exposed, and being able to place electrodes in an exact anatomical location, were considered special advantages. The ability to control the concentration of drug applied to tissue is a crucial advantage of pharmacological characterisation of functional receptors, and the lack of blood-brain barrier will allow the use of a wide range of investigative agents. Electrochemical methods have been used with brain slices, mainly for off line analysis of substances released into perfusion fluids, followed by chromatographic separation and electrochemical detection (see Joseph and Marsden (1986) for a review). Kelly and Wightman (1987) have used carbon fibre microelectrodes to measure dopamine overflow and diffusion in brain slices but they have not measured dopamine release in real time and they did not characterise the biochemistry of the system. Here, we describe in detail the method we have used to measure evoked release of endogenous dopamine and provide pharmacological and electrochemical evidence in support of our conclusions.
Methods
Preparation and incubation of brain slices The brain was removed from a male Wistar-derived A H / A rat (100-180 g) and placed on a pad of filter papers on a raised perspex platform soaked with ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (raM): NaC1 124, KC1 2, KH2PO4 1.25, MgSO4 • 7 H20 2, NaHCO 3 25, D-glucose 11, CaC12 2; the solution was saturated with 95% 02/5% CO 2. Throughout the dissection the required brain area was liberally bathed in ice-cold ACSF. Two coronal cuts, about 6 mm apart, were made to isolate the central part of the striatum, and a block of tissue containing the right striatum was glued using cyanoacrylate adhesive to the cutting stage of an Oxford vibratome. A single 350 ~tm thick slice was taken out at
the widest point, corresponding to the region 9.20-9.70 mm anterior to the interaural line according to the atlas of Paxinos and Watson (1982). This slice was placed on a platinum grid in an incubation chamber based on the design by Richards and Tegg (1977), and held in place with nylon mesh in a plastic frame. The slice was submerged beneath the surface of the ACSF throughout the experiment, and superfused at 0.70 ml. min- 1 using one of 2 Precidor infusion pumps connected by a 3-way tap. A period of 45 min was allowed for recovery before recordings were made.
Fast cyclic ooltarnmetry Improvements and modifications have been introduced for fast cyclic voltammetry (FCV) since originally described (Armstrong-James et al. 1981); those relevant to this set of experiments are briefly described here. A 3-electrode potentiostat circuit was used as recently described by Millar and Barnett (1988). This differs from the conventional electrochemical potentiostat in that potential scan is applied to the working (carbon fibre) electrode, and the auxiliary electrode (the platinum grid of the incubation chamber) is maintained at ground potential. This arrangement of electrodes results in an improved signal-to-noise ratio and enhanced sensitivity. The working electrode was located using a Prior micromanipulator 75 #m below the surface of the slice in a light band of the striatum, as viewed through a binocular microscope with the slice illuminated from below. A bipolar st'tmulating microelectrode (tip separation 100 #m), kindly supplied by Dr. C.D. Riclaards, was placed straddling the same light band, 200-400 #m ventromedial to the working electrode. Constant voltage stimulation and timing was effected using a D4030 Digitimer and DS2 constant voltage stimulator. The applied waveform was 1,5 cycles of a 100Hz triangular ramp, scanning between - 1 . 0 and + 1.4 V relative to the Ag/AgC12 reference electrode at a voltage scan rate of 480 V. s-1 (Fig. la). The scan was applied twice a second and between scans the potential was maintained at 0 V. The signal from the headstage was suitably amplified and fed into a Nicolet 390 digital storage oscilloscope.
39 FAST
CYCLIC
VOLTAMMETR¥
A V + t-41 ~
O
vs 0i- ----~ ~ ~
Ag/AgCI / - t ,0L
,~
B
-587 OximV dation
~" /,~ -213mV Reduction
Biochemicals Inc.), nialamide (Pfizer Ltd.), RO 4-1284 (Roche), tetrodotoxin (Sigma Chemical Co. Ltd.), L-tyrosine HC! (Sigma), G B R 12909 and nialamide were dissolved with the addition of a little 0.1 M HC1. All other drugs were dissolved in distilled water.
Results
Effects of temperature and L-tyrosine Fig. 1. Measurement by fast cyclicvoltammetry.(A) Waveform applied to the working electrode. (B) Waveform produced by the working electrode in response to the applied waveform. The points indicated by arrows show the positions at which changes occur when the tissue is stimulated or the electrode placed in a solution of dopamine. (C) Signal produced by electronic subtraction of the 2 waveforms shown in (B) - the height of the larger peak is used to quantify the amount of dopamine present in the vicinity of the electrode. (D) Fig. 1C can be re-plotted as a current vs. voltage plot - a 'voltammogram'.
The current flowing in the working electrode in the absence or presence of dopamine is illustrated in Fig. lb; if the background signal (lower trace) is electronically subtracted from that obtained either when dopamine has been added to the ACSF or following electrical stimulation of the slice, then a difference signal is observed (Fig. lc). This is shown as an upward directed oxidation peak at + 587 mV (first arrow), and a reduction peak at - 2 1 3 mV). The height of the oxidation peak can be measured and, from a calibration curve, the concentration of dopamine released in a slice can be estimated. A sample and hold device set to monitor the size of the oxidation peak was used to monitor changes in extracellular dopamine concentration between and during electrical stimulations (Millar et al. 1985). The output from the sample and hold was into a Y / t chart recorder. A system for monitoting dopamine by FCV is now commercially available (for details please contact J.M.).
Drugs and solutions D e s m e t h y l i m i p r a m i n e HC1 (Desipramine, Geigy Pharmaceuticals), GBR12909 (Research
Initial experiments were performed at 37°C. Attempts were made to evoke release of dopamine with trains of up to four 0.1-ms rectangular pulses, amplitude 20 V, 250 ms apart. At this temperature, no electrochemical signal could be detected from slices in ACSF which contained no uptake blocker. When the selective dopamine uptake blocker, GBR12909 (10 -6 M), was added to the medium, a signal could be detected initially in response to electrical stimulation, but this response declined rapidly over the next 30-60 min following repeated stimulation. Since it was possible that the decline was due to depletion of releasable dopamine within the brain slice, tyrosine (10 -6 M) was added to the ACSF. This procedure did not prolong the period for which a response could be detected. Reducing the temperature to 3 2 ° C increased the response to electrical stimulation so that a signal could be detected even in the absence of GBR12909. When stimulated every 2 min, this signal, an example of which is shown in Fig. 7a, was reliably evoked for over 9 h (Fig. 2): there was a 15-20% increase on average over the first hour, followed by a further gradual increase. All subsequent experiments were performed at 32 ° C. N o basal release was detected at any temperature.
Stimulus parameters The stimulus parameters applied to the bipolar stimulating electrode were reduced progressively to determine the minimum necessary to produce a detectable release of oxidisable material. It was found that a single 0.1-ms pulse was sufficient. The signal amplitude varied with the applied voltage above a certain threshold (approx. 1-2 V), and a voltage of 20 V was chosen as standard.
40 TIME
COURSE
OF R E L E A S E
I
140
.I ...,t...:~,....4::~
12o
.."J:4^.)-~
100
..4,::':
~::.~.::'.:.....:~:~
60 ,y' n - ~ 40
o 20
~
q Time (hours)
Fig. 2. Time course of evoked, release. R=lcasc of dopamine was evoked with single 0.1-rm pulses of 20 V ampfitude delivered every 2 rain performed at 32 ° C. Mean release of all individual pulses for an entire experiment was calculated and each value then plotted as a pexcxmta~ of that mean. The figure shows the combined results of 3 such experiments. (Each point represents the mean with SEM shown at 30-rain periods for the sake of clarity.)
This was not quite supramaximal voltaBe; higher voltages caused tissue damage around the site of stimulation, possibly due to electrolysis of the ACSF.
Voltammetric identification of the substance released by electrical stimulation The current-voltage plot for the oxidisable 'electroactive' species d~tccted upon dectdcal stimulation was identical to that for exogenous dopamine (Fig. 3a). It differed from the equivalent plots obtained in solutions of other oxidisable neurotransmitters and mctabotitcs which are present in striatum, includin$ noxadtenaline, 5-hydroxytryptamine, DOPAC and I-IVA (Fig. 3C-F). In solutions of exogenous dopamiae, the peak response of the carbon fibre working electrode was approximately linearly proportional to the concentration over the r~n~. 10 -7 M (minimum detectable limit) to 10-5 M.
Evidence for neuronal origin of the released substance Removal of calcium from the ACSF and replacement with malpaosium abolished the electrieally-evokexl voltammetric signal within 10 rain
(Fig. 4a). The response was rapidly re-established on returning to normal ACSF. Tetrodotoxin (10 -7 M) abolished the signal within 20 min (Fig. 4b). On washout, the response returned to about 70% of the control value after 2 h.
Biochemical characterisation of the released substance The fast-acting reserpine-like drug RO 4-1284 reduced the electrochemical signal in a concentration-dependent manner when added to the ACSF over the range 5 × 10 -9 to 5 × 10 -7 M (Fig. 5). In addition, RO 4-1284 caused a concentration-re-
Stimulated release oxidation +587mV reduction - 213mV
Noradrenaline 1pM oxidation +519mV reduction -133mV
DOPAC 1pM oxidation + 454mV reduction - 279mV
Exogenous Dopamine 1HM oxidation +586mV reduction -226mV
5-Hydroxytryptamine lluM oxidation +579mV reduction - 5 3 and -530mV
HVA lpM oxidation +606mV reduction -139mV
Fig. 3. Cyclic voltammograms. (a) cyclicv o l t a ~ a m of the substance releasedby dectricalstimulationin the st.-iatalslice. This appears almost identical to that produced in the pr¢~ence of a known concentration of dopamine (b). It ~ diffex~t in oxidation/reduction potentials and in shape fro~m the neurotransmitters noradrenalln~e (c) and 5-HT (d) and from the metabolites DOPAC (e) and 1-1VA(f).
41
EFFECT OF CALCIUM REMOVAL
Ro4-1284
"~ 140
]-
120
• p.c0.005
100 o~ >
Ca)
•
100
80
m
--
--
C - control
_
=_
40
" "6
20
00
o
o
Control
Zero Ca ++
50
NgTrmal Ca +* 1hr
g_
EFFECT OF TTX
~
C
140
_~ 120
• p¢0.005
0
~ I~'; ~. "5 ae v
p
37
NSM 01051
Application of fast cyclic voltammetry to measurement of electrically evoked dopamine overflow from brain slices in vitro D.R. Bull 1, p. Palij 3, M.J. Sheehan 1, j. Millar 2, J.A. Stamford 3, Z.L. Kruk 3 and P.P.A. H u m p h r e y 1 1 Department of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts (U. K.L and Departments of 2 Physiology and 3 Pharmacology, The London Hospital Medical College, London El 2AD (U.K.) (Received 7 August 1989) (Revised version received 5 December 1989) (Accepted 7 December 1989)
Key words: Fast cyclic voltammetry; Dopamine; Release; Striatum; In vitro Fast cyclic voltammetry at a carbon fibre microelectrode was used to monitor the time course of dopamine overflow in slices of rat corpus striatum incubated in a brain slice chamber. Dopamine release occurred in response to electrical stimulation. Electrochemical, physiological and pharmacological evidence indicates that release of endogenous dopamine can be measured reliably for up to 9 h and that fast cyclic voltammetry can be used in brain slices for quantitative studies of dopamine release in the CNS.
Introduction
Fast cyclic voltammetry (FCV) is an electrochemical detection method which has been applied to quantification of ionophoresis (Armstrong-James et al. 1980, 1981; Kruk et al. 1980; Millar et al. 1981) and to monitoring of changes of the extracellular concentration of dopamine in the central nervous system of the anaesthetised rat (Millar et al. 1985; Stamford et al. 1986a,b) and measurement of ascorbic acid distribution in the brain (Stamford et al. 1984a,b). The advantages of FCV over other voltammetric methods have been reviewed (Armstrong-James et al. 1981; Kruk 1986) and they can be summarised as measuring evoked endogenous dopamine without the addition of MAOI (as opposed to measuring metabolites), having a time resolution of better than 50 ms, and allowing pharmacological mani-
Correspondence: Dr. D.R. Bull, Dept. of Neuropharmacology, Glaxo Group Research Ltd., Ware, Herts SG12 ODP, U.K.
pulations which allow investigation of storage and release of dopamine (Kruk and Stamford 1985; Stamford et al. 1986c), high and low affinity dopamine uptake (Stamford et al. 1984a,b, 1986d) and control of release by autoreceptors (Stamford et al. 1986d,e, 1987). FCV in vivo is not without some limitations; experiments have to be done in anaesthetised animals, the sensitivity of the method does not allow measurement of basal (background) concentrations of dopamine, the range of drugs and experimental manipulation is limited to agents which can readily cross the blood-brain barrier, and generally, it is only possible to study a single dose of a drug in each experiment. As with all stereotaxic methods, the anatomical location of electrodes cannot be known accurately except by post-experimental histological examination. To overcome some of these limitations, and to extend the range of investigations possible using FCV, we have investigated whether FCV could be used in brain slices. The use of brain slices for qualitative and quantitative neurobiology is established, and the
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
38
advantages of brain slices have been extensively reviewed (Kerkut and Wheal 1981; Dingledine 1984). In applying FCV in brain slices, the ability to control accurately the concentration of drug to which the tissue is exposed, and being able to place electrodes in an exact anatomical location, were considered special advantages. The ability to control the concentration of drug applied to tissue is a crucial advantage of pharmacological characterisation of functional receptors, and the lack of blood-brain barrier will allow the use of a wide range of investigative agents. Electrochemical methods have been used with brain slices, mainly for off line analysis of substances released into perfusion fluids, followed by chromatographic separation and electrochemical detection (see Joseph and Marsden (1986) for a review). Kelly and Wightman (1987) have used carbon fibre microelectrodes to measure dopamine overflow and diffusion in brain slices but they have not measured dopamine release in real time and they did not characterise the biochemistry of the system. Here, we describe in detail the method we have used to measure evoked release of endogenous dopamine and provide pharmacological and electrochemical evidence in support of our conclusions.
Methods
Preparation and incubation of brain slices The brain was removed from a male Wistar-derived A H / A rat (100-180 g) and placed on a pad of filter papers on a raised perspex platform soaked with ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (raM): NaC1 124, KC1 2, KH2PO4 1.25, MgSO4 • 7 H20 2, NaHCO 3 25, D-glucose 11, CaC12 2; the solution was saturated with 95% 02/5% CO 2. Throughout the dissection the required brain area was liberally bathed in ice-cold ACSF. Two coronal cuts, about 6 mm apart, were made to isolate the central part of the striatum, and a block of tissue containing the right striatum was glued using cyanoacrylate adhesive to the cutting stage of an Oxford vibratome. A single 350 ~tm thick slice was taken out at
the widest point, corresponding to the region 9.20-9.70 mm anterior to the interaural line according to the atlas of Paxinos and Watson (1982). This slice was placed on a platinum grid in an incubation chamber based on the design by Richards and Tegg (1977), and held in place with nylon mesh in a plastic frame. The slice was submerged beneath the surface of the ACSF throughout the experiment, and superfused at 0.70 ml. min- 1 using one of 2 Precidor infusion pumps connected by a 3-way tap. A period of 45 min was allowed for recovery before recordings were made.
Fast cyclic ooltarnmetry Improvements and modifications have been introduced for fast cyclic voltammetry (FCV) since originally described (Armstrong-James et al. 1981); those relevant to this set of experiments are briefly described here. A 3-electrode potentiostat circuit was used as recently described by Millar and Barnett (1988). This differs from the conventional electrochemical potentiostat in that potential scan is applied to the working (carbon fibre) electrode, and the auxiliary electrode (the platinum grid of the incubation chamber) is maintained at ground potential. This arrangement of electrodes results in an improved signal-to-noise ratio and enhanced sensitivity. The working electrode was located using a Prior micromanipulator 75 #m below the surface of the slice in a light band of the striatum, as viewed through a binocular microscope with the slice illuminated from below. A bipolar st'tmulating microelectrode (tip separation 100 #m), kindly supplied by Dr. C.D. Riclaards, was placed straddling the same light band, 200-400 #m ventromedial to the working electrode. Constant voltage stimulation and timing was effected using a D4030 Digitimer and DS2 constant voltage stimulator. The applied waveform was 1,5 cycles of a 100Hz triangular ramp, scanning between - 1 . 0 and + 1.4 V relative to the Ag/AgC12 reference electrode at a voltage scan rate of 480 V. s-1 (Fig. la). The scan was applied twice a second and between scans the potential was maintained at 0 V. The signal from the headstage was suitably amplified and fed into a Nicolet 390 digital storage oscilloscope.
39 FAST
CYCLIC
VOLTAMMETR¥
A V + t-41 ~
O
vs 0i- ----~ ~ ~
Ag/AgCI / - t ,0L
,~
B
-587 OximV dation
~" /,~ -213mV Reduction
Biochemicals Inc.), nialamide (Pfizer Ltd.), RO 4-1284 (Roche), tetrodotoxin (Sigma Chemical Co. Ltd.), L-tyrosine HC! (Sigma), G B R 12909 and nialamide were dissolved with the addition of a little 0.1 M HC1. All other drugs were dissolved in distilled water.
Results
Effects of temperature and L-tyrosine Fig. 1. Measurement by fast cyclicvoltammetry.(A) Waveform applied to the working electrode. (B) Waveform produced by the working electrode in response to the applied waveform. The points indicated by arrows show the positions at which changes occur when the tissue is stimulated or the electrode placed in a solution of dopamine. (C) Signal produced by electronic subtraction of the 2 waveforms shown in (B) - the height of the larger peak is used to quantify the amount of dopamine present in the vicinity of the electrode. (D) Fig. 1C can be re-plotted as a current vs. voltage plot - a 'voltammogram'.
The current flowing in the working electrode in the absence or presence of dopamine is illustrated in Fig. lb; if the background signal (lower trace) is electronically subtracted from that obtained either when dopamine has been added to the ACSF or following electrical stimulation of the slice, then a difference signal is observed (Fig. lc). This is shown as an upward directed oxidation peak at + 587 mV (first arrow), and a reduction peak at - 2 1 3 mV). The height of the oxidation peak can be measured and, from a calibration curve, the concentration of dopamine released in a slice can be estimated. A sample and hold device set to monitor the size of the oxidation peak was used to monitor changes in extracellular dopamine concentration between and during electrical stimulations (Millar et al. 1985). The output from the sample and hold was into a Y / t chart recorder. A system for monitoting dopamine by FCV is now commercially available (for details please contact J.M.).
Drugs and solutions D e s m e t h y l i m i p r a m i n e HC1 (Desipramine, Geigy Pharmaceuticals), GBR12909 (Research
Initial experiments were performed at 37°C. Attempts were made to evoke release of dopamine with trains of up to four 0.1-ms rectangular pulses, amplitude 20 V, 250 ms apart. At this temperature, no electrochemical signal could be detected from slices in ACSF which contained no uptake blocker. When the selective dopamine uptake blocker, GBR12909 (10 -6 M), was added to the medium, a signal could be detected initially in response to electrical stimulation, but this response declined rapidly over the next 30-60 min following repeated stimulation. Since it was possible that the decline was due to depletion of releasable dopamine within the brain slice, tyrosine (10 -6 M) was added to the ACSF. This procedure did not prolong the period for which a response could be detected. Reducing the temperature to 3 2 ° C increased the response to electrical stimulation so that a signal could be detected even in the absence of GBR12909. When stimulated every 2 min, this signal, an example of which is shown in Fig. 7a, was reliably evoked for over 9 h (Fig. 2): there was a 15-20% increase on average over the first hour, followed by a further gradual increase. All subsequent experiments were performed at 32 ° C. N o basal release was detected at any temperature.
Stimulus parameters The stimulus parameters applied to the bipolar stimulating electrode were reduced progressively to determine the minimum necessary to produce a detectable release of oxidisable material. It was found that a single 0.1-ms pulse was sufficient. The signal amplitude varied with the applied voltage above a certain threshold (approx. 1-2 V), and a voltage of 20 V was chosen as standard.
40 TIME
COURSE
OF R E L E A S E
I
140
.I ...,t...:~,....4::~
12o
.."J:4^.)-~
100
..4,::':
~::.~.::'.:.....:~:~
60 ,y' n - ~ 40
o 20
~
q Time (hours)
Fig. 2. Time course of evoked, release. R=lcasc of dopamine was evoked with single 0.1-rm pulses of 20 V ampfitude delivered every 2 rain performed at 32 ° C. Mean release of all individual pulses for an entire experiment was calculated and each value then plotted as a pexcxmta~ of that mean. The figure shows the combined results of 3 such experiments. (Each point represents the mean with SEM shown at 30-rain periods for the sake of clarity.)
This was not quite supramaximal voltaBe; higher voltages caused tissue damage around the site of stimulation, possibly due to electrolysis of the ACSF.
Voltammetric identification of the substance released by electrical stimulation The current-voltage plot for the oxidisable 'electroactive' species d~tccted upon dectdcal stimulation was identical to that for exogenous dopamine (Fig. 3a). It differed from the equivalent plots obtained in solutions of other oxidisable neurotransmitters and mctabotitcs which are present in striatum, includin$ noxadtenaline, 5-hydroxytryptamine, DOPAC and I-IVA (Fig. 3C-F). In solutions of exogenous dopamiae, the peak response of the carbon fibre working electrode was approximately linearly proportional to the concentration over the r~n~. 10 -7 M (minimum detectable limit) to 10-5 M.
Evidence for neuronal origin of the released substance Removal of calcium from the ACSF and replacement with malpaosium abolished the electrieally-evokexl voltammetric signal within 10 rain
(Fig. 4a). The response was rapidly re-established on returning to normal ACSF. Tetrodotoxin (10 -7 M) abolished the signal within 20 min (Fig. 4b). On washout, the response returned to about 70% of the control value after 2 h.
Biochemical characterisation of the released substance The fast-acting reserpine-like drug RO 4-1284 reduced the electrochemical signal in a concentration-dependent manner when added to the ACSF over the range 5 × 10 -9 to 5 × 10 -7 M (Fig. 5). In addition, RO 4-1284 caused a concentration-re-
Stimulated release oxidation +587mV reduction - 213mV
Noradrenaline 1pM oxidation +519mV reduction -133mV
DOPAC 1pM oxidation + 454mV reduction - 279mV
Exogenous Dopamine 1HM oxidation +586mV reduction -226mV
5-Hydroxytryptamine lluM oxidation +579mV reduction - 5 3 and -530mV
HVA lpM oxidation +606mV reduction -139mV
Fig. 3. Cyclic voltammograms. (a) cyclicv o l t a ~ a m of the substance releasedby dectricalstimulationin the st.-iatalslice. This appears almost identical to that produced in the pr¢~ence of a known concentration of dopamine (b). It ~ diffex~t in oxidation/reduction potentials and in shape fro~m the neurotransmitters noradrenalln~e (c) and 5-HT (d) and from the metabolites DOPAC (e) and 1-1VA(f).
41
EFFECT OF CALCIUM REMOVAL
Ro4-1284
"~ 140
]-
120
• p.c0.005
100 o~ >
Ca)
•
100
80
m
--
--
C - control
_
=_
40
" "6
20
00
o
o
Control
Zero Ca ++
50
NgTrmal Ca +* 1hr
g_
EFFECT OF TTX
~
C
140
_~ 120
• p¢0.005
0
~ I~'; ~. "5 ae v
p
