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In vivo electrochemistry - principles and applications
Life Sciences, Vol. 41, pp. 865-868 Printed in the U.S.A.
Pergamon JourDals
IN VlVO ELECTROCHEMISTRY
-
PRINCIPLES AND APPLICATIONS
Keith F. Martin and Charles A. Marsden Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.
Summary Differential pulse voltammetry (DPV) with electrically pretreated carbon fibre microelectrodes has been used to monitor dopamine (DA) and serotonin (NHT) metabolism in specific regions of the rat brain in vivo. Using this technique we have located the 5HT receptor subtype responsible for controlling 5HT release and metabolism in the suprachiasmatic nucleus. The autoreceptor involved in the control of DA metabolism has also been studied and the effects of chronic neuroleptic administration on their sensitivity determined. There has been increasing interest during the last few years in the application of electrochemical methodology to the development of in vivo biosensors. An area where electrochemical technology can make an impact is in the development of biosensors for specific neurotransmitters and their metabolites to determine the selectivity of drugs for specific neuronal systems in the brain. It is essential that the specificity of action is demonstrated in vivo as well as using in vitro tests. The objective of this paper is to show how in vivo voltammetry with carbon fibre micro-electrodes can monitor rapid changes in the metabolism and release of amine neurotransmitters in specific brain areas. This technique is now providing new information about the regulation of neuronal activity in the brain and can aid the development of selective drugs for the treatment of neurological and psychiatric disease. Principles The principles of in vivo voltammetry have been presented in detail elsewhere (I), and therefore will only be dealt with briefly here. In vivo voltammetry makes use of a well-known problem for those working with cateehol- and indoleamines - their ease of oxidation (Figure I). This reaction takes place on the surface of a carbon electrode which acts as the oxidising agent. Most neuroscientists are now familiar with the first practical application of electrochemistry, namely the use of an electrochemical detector combined with high performance liquid chromatography. Essentially, the in vivo technique involves the implantation of a miniaturised version of the electrochemical detector into the brain. However, in vivo there is no chromatographic separation of the compounds in the extracellular fluid, therefore one has to rely upon the ability of the voltammetric techniques to distinguish between the various compounds on the basis of the different potentials at which these compounds oxidise (Figure 1). These oxidative reactions occur at the surface of the working electrode and the electrons generated are proportional to the number of molecules oxidised. The form of the electrochemical signal monitored depends upon the measurement 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
In V~Uo Voltammetry
866
Vol. 41, No, 7, 1987
technique employed and the nature of the working electrode.
In®leamine --R O ~ ~ _
HO~ ~ N
R +2e÷2H
H Oopamine OOPAC
Endogenous Amines
5HT 5HIAA
HVA
Tryotomine
!
o Others
T Ascorbic
Uric acid
÷I,0
Trypfophan
acid FIGURE 1. (Top) Oxidation of indoleamines to form an orthoquinone and (bottom) a diagram to indicate the approximate oxidation potentials of some of the compounds found in the extraeellular space. Measurement techniques. Essentially there are two approaches. In the first method, known as chronoamperometry, a square wave potential is applied to the working electrode for a fixed time (e.g. +0.5 V for I s) and the current generated during the last 10% of the fixed time measured thus avoiding the inclusion of the capacitance current in the measurement. All compounds which oxidise at or below the applied potential are oxidised and therefore this technique provides quantitative but not qualitative information. Its main advantage is the speed at which measurements can be made. The second method is to apply a steadily increasing ramp potential, of a pre-determined range, to the working electrode. At the oxidation potential of a compound there will be a peak in the current recorded and providing there is a reasonable difference between the oxidation potentials of two or more compounds (e.g. 150 mV) they will produce separate oxidation peaks. Therefore qualitative as well as quantitative data can be obtained. Peak resolution can be improved by using modifications of the basic linear ramp technique, for example, by super-imposing regular step potentials (of e.g. 30 ms duration, 50 mV amplitude, 2 Hz) on the basic linear ramp. This technique is known as differential pulse voltammetry (DFV) and is the one used routinely in our laboratory. Working electrode. Because the electrochemical signals recorded in vivo are in the nano-amp range the working electrode needs to be made of material with a very low residual current. This has therefore limited electrode construction to carbon based materials. In addition, the working electrode must be able to distinguish between compounds with similar oxidation potentials which are found in the extraeellular fluid. For example, a considerable problem was posed by the high levels of aseorbic acid in the brain (2) which oxidise at a similar potential to that of dopamine and its metabolite DOPAC. In addition the indoleamines oxidise at a similar potential
Vol. 41, No. 7, 1987
In V~Vo Voltammetry
867
to uric acid, which is also found in high concentrations in the brain (3). However, using carbon fibre electrodes oxidation of uric acid accounts for a maximum of 30~ of the indole oxidation peak (4). Finally, since ultimately we are interested in neurotransmitter release, we would also like an electrode to distinguish between the acid metabolites and the parent amines, dopamine and SHT. This latter problem has some way to go before it is fully resolved and is in ~art due to the very low extracellular concentrations of the amines (NxlO-SM) compared to the metabolites (5xlO-6M). The former problems with ascorbic acid have been largely overcome (5). Oxidation of SHIAA can also be separated from that of ascorbic acid and DOPAC (6). The working electrodes used in our laboratory (7) are made from three pyrolytic carbon fibres (each $ ~m diameter) inserted into a glass pipette pulled to a fine tip. The tip is sealed and the fibres trimmed to 300 ~m length. Electrical contact with the fibres is made using electrical conductive paint and silver wire and the joint strengthened with polyester resin. These working electrodes are used with a silver/silver chloride reference electrode and a silver auxiliary electrode. Electrochemical separation between compounds is obtained by electrically pre-treating these electrodes prior to implantation. Full details are given in Sharp et al (8). In our hands these electrodes remain stable for up to 8 h. These carbon fibre electrodes also have the advantage that the tip is only 20 ~m in diameter and are therefore ideally suited to recording from small brain regions such as the suprachiasmatic nuclei (SCN). With anaesthetised animals the choice of anaesthetic agent is important. Recent data from our laboratory has shown that halothane provides a stable base-line and drug induced responses similar to those observed in the freely moving animal (9). Applications We have placed voltammetric electrodes in one striatum or nuc. accumbeus and an intracerebral dialysis loop, perfused with physiological saline (I pi/min) in the contralateral striatum. Levels of the amine metabolites are measured in 20 min dialysis perfusates using HPLC with electrochemical detection. In the striatum and nuc. accumbens large DOPAC oxidation peaks are observed and changes in the size of this peak following drug administration correspond with changes in the levels of DOPAC measured in the dialysis samples. Thus d-amphetamine (2 mg/kg) decreases the voltammetric DOPAC peak by 45~ and DOPAC in the dialysis samples by 58% while the neuroleptic drug haloperidol (0.5 mg/kg) increased the peak by 118~ and DOPAC levels by 113%. There is a similar good correlation between the decrease in 5HIAA measured by these techniques following the MAO inhibitor tranylcypromine (10 mg/kg i.p.). These results support evidence that our in vivo electrodes monitor changes in amine metabolites. However, administration of 5-hydroxytryptophan (SHTP) results in a 97~ increase in the voltammetric signal whilst 5HIAA in dialysis perfusates increases 447~ suggesting that there may be another component to the 5HIAA peak. One possible candidate is uric acid since injection of uricase around the surface of the electrode in vivo reduces the peak height (-30~) (4). More direct assessment of the effects of neuroleptic drugs on selected dopaminergic systems has been obtained by micro-infusing haloperidol (2.5 pg) or dopamine (100 ~g) onto the cell bodies in the ventral tegmental area and recording changes in DOPAC in the nuc. accumbens terminal area. Results show haloperidol increases while apomorphine decreases metabolism of dopamine in these neurones supporting evidence for autoreceptor control of the dopamine mesolimbic systems (I0). With electrodes placed into both the striatum and nuc. accumbens we have compared the effects of atypical neuroleptics, after
868
In Vivo Voltammetry
Vol. 41, No.
acute and chronic administration, and neurotensin on the nigrostriatal mesolimbic dopamine pathways (11,12).
7, 1987
and
There is in vitro evidence that 5HT neuronal activity is under autoreceptor control and we have shown that administration of the 5HT 1 receptor agonist 5-methoxy-S(1,2,S,6-tetrahydropyridine-4-yl) H-indole (RU 24969) (10 mg/kg i.p.) decreases the 5HIAA oxidation peak in the pre-frontal cortex and the SCN (13,14, Figure 2). A similar decrease is observed using intracerebral dialysis (performed simultaneously with voltammetry) together with a reduction in 5HT levels. These results support the view that 5HT I receptors are involved in the autoregulation of 5HT release and metabolism. By using local injection techniques we have now established that these autoreceptors are of the 5HTIB type and located on the nerve terminals (15,16). RU2,~%9 (lOm~/k 9 i. p,
FIGURE 2. Typical voltammograms obtained from the SCN in an experiment to study the effect of i.p. administration of the 5HT 1 receptor agonist RU 24969 (10 mg/kg). The vertical bar represents 1 nA. In conclusion, in vivo voltammetry is a rapid and sensitive method for monitoring the selectivity of drug action on dopamine and 5HT receptors in vivo. We thank the Welleome Trust and the MRC for financial
support.
References I.
Z. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
R.N. ADAMS and C.A. MARSDEN, Handbook of Psychopharmacology, (eds. L.L. IVERSEN, S. IVERSEN and S.H. SNYDER) pp. 1-74, Plenum Press, New York (1982) I.N. MEFFORD, A.F. OKE and R.N. ADAMS, Brain Res. Z12 223-226 (1981) T. ZETTERSTROM, T. SHARP, C.A. MARSDEN and U. UNGERSTEDT, J. Neurochem. 41 1769-1773 (1983) F. CRESPI, T. SHARP, N.T. MAIDMENT and C.A. MARSDEN, Neurosci. Letters 4_38 203-208 (1983) F. GONON, M. BUDA and J.F. PUJOL, Measurement of Neurotransmitter Release In Vivo, (ed. C.A. MARSDEN) pp. 153-172, J. Wiley and Son, Chichester (19~4) R. CESPUGLIO, H. FARADJI, Z. HAHN and M. JOUVET, i.b.i.d. pp. 173-192 (1984) C.A. MARSDEN and K . F . MARTIN, Br. J . Pharmac. 8 9 , 277-286 (1986) T. SHARP, N.T. MAIDMENT, H . P . BRAZELL, T. ZETTERSTROM, G.W. BENNETT and C.A. MARSDEN, N e u r o s c i e n c e 1 2 1 2 1 3 - 1 2 2 1 (1984) A.P.D.W. FORD and C.A. MARSDEN, B r a i n Res. 3 7 6 1 6 2 - 1 6 6 (1986) N.T. MAIDMENT and C.A. MARSDEN, B r a i n Res. 338, 317-325 (1985) N.T. MAIDMENT and C.A. MARSDEN, N e u r o p h a r m a e . 2_66187-194 (1987) N.T. MAIDMENT and C.A. MARSDEN, Eur. J . Pharmac. (1987) I n P r e s s M.P. BRAZELL, C.A. MARSDEN, A.P. NISBET and C. ROUTLEDGE Br. J . P h a r m a c . 8_66209-216 (1985) K . F . MARTIN and C.A. MARSDEN, E u r . J . P h a r m a e o l . 1 2 1 1 3 5 - 1 4 0 (1986) C.A. MARSDEN and K . F . MARTIN, Br. J . P h a r m a e . 8 5 2 1 9 P (1985) C.A. MARSDEN and K . F . MARTIN, Br. J . P h a r m a e . 8 5 4 4 5 P (1985)
Pergamon JourDals
IN VlVO ELECTROCHEMISTRY
-
PRINCIPLES AND APPLICATIONS
Keith F. Martin and Charles A. Marsden Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.
Summary Differential pulse voltammetry (DPV) with electrically pretreated carbon fibre microelectrodes has been used to monitor dopamine (DA) and serotonin (NHT) metabolism in specific regions of the rat brain in vivo. Using this technique we have located the 5HT receptor subtype responsible for controlling 5HT release and metabolism in the suprachiasmatic nucleus. The autoreceptor involved in the control of DA metabolism has also been studied and the effects of chronic neuroleptic administration on their sensitivity determined. There has been increasing interest during the last few years in the application of electrochemical methodology to the development of in vivo biosensors. An area where electrochemical technology can make an impact is in the development of biosensors for specific neurotransmitters and their metabolites to determine the selectivity of drugs for specific neuronal systems in the brain. It is essential that the specificity of action is demonstrated in vivo as well as using in vitro tests. The objective of this paper is to show how in vivo voltammetry with carbon fibre micro-electrodes can monitor rapid changes in the metabolism and release of amine neurotransmitters in specific brain areas. This technique is now providing new information about the regulation of neuronal activity in the brain and can aid the development of selective drugs for the treatment of neurological and psychiatric disease. Principles The principles of in vivo voltammetry have been presented in detail elsewhere (I), and therefore will only be dealt with briefly here. In vivo voltammetry makes use of a well-known problem for those working with cateehol- and indoleamines - their ease of oxidation (Figure I). This reaction takes place on the surface of a carbon electrode which acts as the oxidising agent. Most neuroscientists are now familiar with the first practical application of electrochemistry, namely the use of an electrochemical detector combined with high performance liquid chromatography. Essentially, the in vivo technique involves the implantation of a miniaturised version of the electrochemical detector into the brain. However, in vivo there is no chromatographic separation of the compounds in the extracellular fluid, therefore one has to rely upon the ability of the voltammetric techniques to distinguish between the various compounds on the basis of the different potentials at which these compounds oxidise (Figure 1). These oxidative reactions occur at the surface of the working electrode and the electrons generated are proportional to the number of molecules oxidised. The form of the electrochemical signal monitored depends upon the measurement 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
In V~Uo Voltammetry
866
Vol. 41, No, 7, 1987
technique employed and the nature of the working electrode.
In®leamine --R O ~ ~ _
HO~ ~ N
R +2e÷2H
H Oopamine OOPAC
Endogenous Amines
5HT 5HIAA
HVA
Tryotomine
!
o Others
T Ascorbic
Uric acid
÷I,0
Trypfophan
acid FIGURE 1. (Top) Oxidation of indoleamines to form an orthoquinone and (bottom) a diagram to indicate the approximate oxidation potentials of some of the compounds found in the extraeellular space. Measurement techniques. Essentially there are two approaches. In the first method, known as chronoamperometry, a square wave potential is applied to the working electrode for a fixed time (e.g. +0.5 V for I s) and the current generated during the last 10% of the fixed time measured thus avoiding the inclusion of the capacitance current in the measurement. All compounds which oxidise at or below the applied potential are oxidised and therefore this technique provides quantitative but not qualitative information. Its main advantage is the speed at which measurements can be made. The second method is to apply a steadily increasing ramp potential, of a pre-determined range, to the working electrode. At the oxidation potential of a compound there will be a peak in the current recorded and providing there is a reasonable difference between the oxidation potentials of two or more compounds (e.g. 150 mV) they will produce separate oxidation peaks. Therefore qualitative as well as quantitative data can be obtained. Peak resolution can be improved by using modifications of the basic linear ramp technique, for example, by super-imposing regular step potentials (of e.g. 30 ms duration, 50 mV amplitude, 2 Hz) on the basic linear ramp. This technique is known as differential pulse voltammetry (DFV) and is the one used routinely in our laboratory. Working electrode. Because the electrochemical signals recorded in vivo are in the nano-amp range the working electrode needs to be made of material with a very low residual current. This has therefore limited electrode construction to carbon based materials. In addition, the working electrode must be able to distinguish between compounds with similar oxidation potentials which are found in the extraeellular fluid. For example, a considerable problem was posed by the high levels of aseorbic acid in the brain (2) which oxidise at a similar potential to that of dopamine and its metabolite DOPAC. In addition the indoleamines oxidise at a similar potential
Vol. 41, No. 7, 1987
In V~Vo Voltammetry
867
to uric acid, which is also found in high concentrations in the brain (3). However, using carbon fibre electrodes oxidation of uric acid accounts for a maximum of 30~ of the indole oxidation peak (4). Finally, since ultimately we are interested in neurotransmitter release, we would also like an electrode to distinguish between the acid metabolites and the parent amines, dopamine and SHT. This latter problem has some way to go before it is fully resolved and is in ~art due to the very low extracellular concentrations of the amines (NxlO-SM) compared to the metabolites (5xlO-6M). The former problems with ascorbic acid have been largely overcome (5). Oxidation of SHIAA can also be separated from that of ascorbic acid and DOPAC (6). The working electrodes used in our laboratory (7) are made from three pyrolytic carbon fibres (each $ ~m diameter) inserted into a glass pipette pulled to a fine tip. The tip is sealed and the fibres trimmed to 300 ~m length. Electrical contact with the fibres is made using electrical conductive paint and silver wire and the joint strengthened with polyester resin. These working electrodes are used with a silver/silver chloride reference electrode and a silver auxiliary electrode. Electrochemical separation between compounds is obtained by electrically pre-treating these electrodes prior to implantation. Full details are given in Sharp et al (8). In our hands these electrodes remain stable for up to 8 h. These carbon fibre electrodes also have the advantage that the tip is only 20 ~m in diameter and are therefore ideally suited to recording from small brain regions such as the suprachiasmatic nuclei (SCN). With anaesthetised animals the choice of anaesthetic agent is important. Recent data from our laboratory has shown that halothane provides a stable base-line and drug induced responses similar to those observed in the freely moving animal (9). Applications We have placed voltammetric electrodes in one striatum or nuc. accumbeus and an intracerebral dialysis loop, perfused with physiological saline (I pi/min) in the contralateral striatum. Levels of the amine metabolites are measured in 20 min dialysis perfusates using HPLC with electrochemical detection. In the striatum and nuc. accumbens large DOPAC oxidation peaks are observed and changes in the size of this peak following drug administration correspond with changes in the levels of DOPAC measured in the dialysis samples. Thus d-amphetamine (2 mg/kg) decreases the voltammetric DOPAC peak by 45~ and DOPAC in the dialysis samples by 58% while the neuroleptic drug haloperidol (0.5 mg/kg) increased the peak by 118~ and DOPAC levels by 113%. There is a similar good correlation between the decrease in 5HIAA measured by these techniques following the MAO inhibitor tranylcypromine (10 mg/kg i.p.). These results support evidence that our in vivo electrodes monitor changes in amine metabolites. However, administration of 5-hydroxytryptophan (SHTP) results in a 97~ increase in the voltammetric signal whilst 5HIAA in dialysis perfusates increases 447~ suggesting that there may be another component to the 5HIAA peak. One possible candidate is uric acid since injection of uricase around the surface of the electrode in vivo reduces the peak height (-30~) (4). More direct assessment of the effects of neuroleptic drugs on selected dopaminergic systems has been obtained by micro-infusing haloperidol (2.5 pg) or dopamine (100 ~g) onto the cell bodies in the ventral tegmental area and recording changes in DOPAC in the nuc. accumbens terminal area. Results show haloperidol increases while apomorphine decreases metabolism of dopamine in these neurones supporting evidence for autoreceptor control of the dopamine mesolimbic systems (I0). With electrodes placed into both the striatum and nuc. accumbens we have compared the effects of atypical neuroleptics, after
868
In Vivo Voltammetry
Vol. 41, No.
acute and chronic administration, and neurotensin on the nigrostriatal mesolimbic dopamine pathways (11,12).
7, 1987
and
There is in vitro evidence that 5HT neuronal activity is under autoreceptor control and we have shown that administration of the 5HT 1 receptor agonist 5-methoxy-S(1,2,S,6-tetrahydropyridine-4-yl) H-indole (RU 24969) (10 mg/kg i.p.) decreases the 5HIAA oxidation peak in the pre-frontal cortex and the SCN (13,14, Figure 2). A similar decrease is observed using intracerebral dialysis (performed simultaneously with voltammetry) together with a reduction in 5HT levels. These results support the view that 5HT I receptors are involved in the autoregulation of 5HT release and metabolism. By using local injection techniques we have now established that these autoreceptors are of the 5HTIB type and located on the nerve terminals (15,16). RU2,~%9 (lOm~/k 9 i. p,
FIGURE 2. Typical voltammograms obtained from the SCN in an experiment to study the effect of i.p. administration of the 5HT 1 receptor agonist RU 24969 (10 mg/kg). The vertical bar represents 1 nA. In conclusion, in vivo voltammetry is a rapid and sensitive method for monitoring the selectivity of drug action on dopamine and 5HT receptors in vivo. We thank the Welleome Trust and the MRC for financial
support.
References I.
Z. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
R.N. ADAMS and C.A. MARSDEN, Handbook of Psychopharmacology, (eds. L.L. IVERSEN, S. IVERSEN and S.H. SNYDER) pp. 1-74, Plenum Press, New York (1982) I.N. MEFFORD, A.F. OKE and R.N. ADAMS, Brain Res. Z12 223-226 (1981) T. ZETTERSTROM, T. SHARP, C.A. MARSDEN and U. UNGERSTEDT, J. Neurochem. 41 1769-1773 (1983) F. CRESPI, T. SHARP, N.T. MAIDMENT and C.A. MARSDEN, Neurosci. Letters 4_38 203-208 (1983) F. GONON, M. BUDA and J.F. PUJOL, Measurement of Neurotransmitter Release In Vivo, (ed. C.A. MARSDEN) pp. 153-172, J. Wiley and Son, Chichester (19~4) R. CESPUGLIO, H. FARADJI, Z. HAHN and M. JOUVET, i.b.i.d. pp. 173-192 (1984) C.A. MARSDEN and K . F . MARTIN, Br. J . Pharmac. 8 9 , 277-286 (1986) T. SHARP, N.T. MAIDMENT, H . P . BRAZELL, T. ZETTERSTROM, G.W. BENNETT and C.A. MARSDEN, N e u r o s c i e n c e 1 2 1 2 1 3 - 1 2 2 1 (1984) A.P.D.W. FORD and C.A. MARSDEN, B r a i n Res. 3 7 6 1 6 2 - 1 6 6 (1986) N.T. MAIDMENT and C.A. MARSDEN, B r a i n Res. 338, 317-325 (1985) N.T. MAIDMENT and C.A. MARSDEN, N e u r o p h a r m a e . 2_66187-194 (1987) N.T. MAIDMENT and C.A. MARSDEN, Eur. J . Pharmac. (1987) I n P r e s s M.P. BRAZELL, C.A. MARSDEN, A.P. NISBET and C. ROUTLEDGE Br. J . P h a r m a c . 8_66209-216 (1985) K . F . MARTIN and C.A. MARSDEN, E u r . J . P h a r m a e o l . 1 2 1 1 3 5 - 1 4 0 (1986) C.A. MARSDEN and K . F . MARTIN, Br. J . P h a r m a e . 8 5 2 1 9 P (1985) C.A. MARSDEN and K . F . MARTIN, Br. J . P h a r m a e . 8 5 4 4 5 P (1985)