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Voltammetry in brain tissue — a new neurophysiological measurement
Brain Research, 55 (1973) 209-213 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
209
Voltammetry in brain tissue - a new neurophysiological measurement
PETER T. KISSINGER*, JONATHAN B. HART** AND RALPH N. ADAMS Department of Chemistry, University of Kansas, Lawrence, Kans. 66044 (U.S.A.} (Accepted February 26th, 1973)
A new dimension would be added to neurophysiology if one could implant a sensor into CNS tissue which would respond to concentrations of neurotransmitter substances. Whether or not such a sensor responds in the classical neurological sense (i.e., directly detecting transmitter release upon nerve pathway stimulation) is actually 'frosting on the cake'. Even a direct reading system that would map steady-state concentrations of endogenous neurotransmitters and related small molecular weight materials in living brain tissue would be of great value. At present such data are inaccessible. Sacrifice of an animal followed by conventional analysis of tissue samples provides results which are always uncertain relative to the living system. Our interests in the electrochemistry of catecholamines, ascorbate and other easily oxidized molecules led us to attempt to monitor these substances in CNS tissue. A first step in evolution of an in vivo probe has been accomplished by successfully mating the techniques of electroanalytical chemistry and neurophysiology. The results reported herein are modest and quantitatively uncertain at present, but we believe they are conceptually important and may stimulate others toward practical developments. The standard electrophysiological experiment uses high input impedance devices to measure the potential of a microelectrode with the express desire of drawing little or no current during the measurement. In contrast to these essentially potentiometric measurements, the dynamic electroanalytical techniques monitor finite current. A small (0 ± 1 V) linearly varying potential is applied between a microelectrode and the medium and the resulting current-potential curve or voltammogram is recorded. The voltammogram is characteristic of the nature and concentration of electro-oxidizable or reducible materials close to the electrode surface. Alternatively, a constant potential may be employed and the current monitored as a function of time (i.e., 'chronoamperometry') as the concentration near the surface changes. Reviews of these completely conventional electroanalytical techniques, with special emphasis on solid microelec* Present address: Department of Chemistry, Michigan State University, East Lansing, Mich. 48823, U.S.A. ** Present address: Haight-Asbury Free Medical Clinic, San Francisco, California, U.S.A.
210
SHORT COMMUNICATIONS
trodes and compounds of pharmacological and neurochemical interest are available in the literature~,a, 6. The only familiar application of finite-current electrochemistry to in vivo measurements is the monitoring of oxygen tension with miniaturized Clark-type membrane electrodes5. Recently, Koryta et aL have employed voltammetric measurements at small platinum electrodes to follow cysteine levels in rat kidney and blood10,11. Carbon paste electrodes were constructed from teflon tubing. A press fit stainless steel insert recessed from one end formed a well into which the paste was packed. Electrodes of this very simple design have been manufactured with active-surface dianaeters from about 50 pm to 1.6 mm and details will be presented elsewhere 9. The carbon paste is formulated by mixing oil (Nujol) and graphite (Ultra Microcrystal Grade Graphite Powder UCP-I-M obtained from the Ultra Carbon Corporation) in a ratio of 2 ml oil to 3 g carbon. Conventional Ag/AgC1 (3 M NaC1) reference electrodes in micropipettes were used as were the exposed ends of teflon-coated silver wire (Medwire Corp.) imbedded directly in tissue. Stainless steel, carbon or platinum auxiliary electrodes were equally satisfactory. For in vivo experiments the arrangement depicted in Fig. I proved to be useful, where the chronically implanted stainless steel cannula (typically 18-gauge) served both as a receptacle for the carbon paste electrode and as the auxiliary electrode. Conventional three-electrode instrumentation was constructed from integrated circuit operational amplifiers (details available on request to P.T.K.). Although the Rm
Wm
~
STAINLESSSTEEL
L
TEFLON CARBONPASTE
Fig. 1. Apparatus for voltammetricmeasurementsin brain.
SHORT COMMUNICATIONS
211
CATHODIC (REDUCTION) CURRENT
E vs AglAg CI +0.6 I
,
+0.4 I
,
.~//-~dSCA~ +0.2 I
_
-0.2
_
5 ~
ANODIC
~,.~ (OXIDATION)
~
lo z~
CURRENT
]
-15
Fig. 2. Cyclicvoltammogram recorded from a carbon paste electrode positioned in the caudate nucleus of an anesthetized rat. Apparatus as per Fig. 1.
objectives are different, this circuitry is perfectly analogous to the conventional voltage clamp apparatus in using negative feedback to control a potential difference and measure a current. Fig. 2 illustrates a typical cyclic voltammogram (3 linear cycles of potential from approx. ---0.2 to + 0 . 6 V) recorded with an electrode implanted in the caudate nucleus of an anesthetized Sprague-Dawley rat. Qualitatively similar voltammograms were obtained with electrode placements in cerebral cortex, hippocampus and a variety of other CNS structures. The salient features of this result include the fact that the oxidation reaction does not yield a stable species reducible in the same potential regime (i.e., the voltammogram is said to be 'irreversible'). This is indicated by the lack of reducing current on the reverse half-cycle. Secondly, the dramatic decrease in response from one cycle to the next suggests rapid depletion of the active species in the neighborhood of the electrode. If one waits (either at open circuit or at 0.0 V) for approx. 100 sec before repeating each successive cycle, then the original response is restored. This may be explained by the presence of a thin intercellular solution of low viscosity adjacent to the electrode surface in equilibrium with the surrounding tissue. A small molecule depleted from this region by the electrode will then be refurnished relatively slowly by diffusion from bulk tissue. While we had hoped to detect dopamine (DA) and/or norepinephrine (NE) directly, unfortunately these substances are oxidized at physiological pH at approximately the same potential as ascorbate. It seems probable that the species most likely to account for the voltammogram shown in Fig. 2 is ascorbic acid. This molecule exhibits similar behavior in vitro and is known to be present in CNS at an average level of about 0.3~0.4 mg/g wet tissue 4,12, which is far in excess of that of the catecholamines z,s.
212
SHORT COMMUNICATIONS
However, because of the heterogeneity of the brain, average levels are of questioJ~able utility in this instance. This is particularly true for the catecholamines. F~r example, the DA level in microscopic varicosities in the striatum has been estimated as high as 8000 ng/g 7. The dimensions of these varicosities are, of course, many orders of magnitude below those of the electrodes employed here. Nevertheless, it is encouraging to note that the concentrations of catecholamines encountered even over larger tissue segments are well within the range detectable by modern electrochemical techniquesZ,7, s. It is important to recognize the possible pitfalls of voltammetry in a medium as complex as brain tissue. The heterogeneity of the sample makes the active electrode area uncertain and complicates diffusion of small molecules to the surface. The ability of voltammetry to resolve species with similar redox properties is really quite poor and a single wave may represent several species. Similarly, electroactive material of relatively high concentration (e.g., ascorbic acid) may swamp out the response to minor components (e.g., dopamine) of equal or greater interest. These facts suggest that a completely satisfactory electrode will require some means of controlling geometry and selectivity. Imaginative use of membranes and, perhaps, immobilized enzymes should prov6 helpful ih this regard. Preliminary experiments with electrodes confined behind a dialysis membrane have shown great promise for in vitro experiments, but this approach has been difficult to implement reproducibly in very small electrodes. Ascorbic acid oxidase isolated from squash has been used to rapidly remove ascorbic acid from solutions and it may be possible to make use of this chemistry in an in vivo probe for adrenergic transmitters. Another fundamental concern in developing in vivo probes is the question of disturbing the system with the measuring device. The response of tissue to injury by the probe may be upsetting, at least for short times. These problems are common to any electrode insertion in CNS tissue. The voltammetric currents passed in the experiments described herein should not be any problem since they are in the nanoampere range. This current is considerably below that ordinarily used for physiological stimulation. Despite the concerns described above, the experimental realization that ordinary voltammetry can be carried out in living brain tissue and gives reasonable results is very exciting. Indeed, if development of the present technique succeeds only in providing an in vivo mapping of discrete ascorbate levels, it will be of considerable significance. Our continuing studies are centered on the technological development of smaller electrodes with greater electrochemical selectivity. The support of this work by the National Institutes of Health via Grant 5 R01 NS08740 is gratefully acknowledged. N O T E A D D E D IN P R O O F
We have recently been able to show that this voltammetry technique can be used to reproducibly and quantitatively monitor the concentration-time course of
SHORT COMMUNICATIONS
213
materials injected into ventricle or caudate nucleus tissue. These materials include ascorbate, dopamine, 6-hydroxydopamine (6-OHDA) and, presumably, any electroactive drug system. This style of measurement is of particular significance to neuropharmacological manipulation studies in small animals. Its particular importance to the study of the interaction of 6-OHDA with neural tissue, which at present is still poorly understood, will be presented in the near future (R. McCreery, R. Dreiling and R. N. Adams, unpublished data).
1 ADAM, H. M., The topology of the brain amines - - a review. In G. HOOPER (Ed.), Metabolism of Amines in the Brain, Macmillan, London, 1971, pp. 4-5. 2 ADAMS,R. N., Applications of modern electroanalytical techniques to pharmaceutical chemistry, J. pharm. Sci., 58 (1969) 1171-1181. 3 ADAMS,R. N., MURRILL, E., MCCREERY, R., BLANK,L., AND KAROLCZAK, M., 6-Hydroxydopamine, a new oxidation mechanism, Europ. J. Pharmacol., 17 (1972) 287-292. 4 ALLISON, J. H., AND STEWART, M. A., Quantitative analysis of ascorbic acid in tissues by gasliquid chromatography, Analyt. Biochem., 43 (1971) 401-409. 5 BlCrtER, H. I., AND KNISELY, M. H., Brain tissue reoxygenation time, demonstrated with a new ultamicro oxygen electrode, J. appl. Physiol., 28 (1970) 387-390. 6 HAWLEY,D., TATWAWADI,S. V., PIEKARSKI,S., AND ADAMS,R. N., Electrochemical studies of the oxidation pathways of catecholamines, J. A mer. chem. Soc., 89 (1967) 447-450. 7 HORNYKIEWICZ,O., Dopamine: Its physiology, pharmacology and pathological neurochemistry. In J. H. BIEL AND L. G. ABOOD (Eds.), Biogenic Amines and Physiological Membranes in Drug Therapy, Vol. 5, Part B, Medical Research Series, Marcel Dekker, New York, 1971, p. 192. 8 IVERSEN, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge University Press, London, 1967, p. 34. 9 KISSINGER,P. T., DREILING,R., KAROLCZAK,M., ANDADAMS,R. N., Semi-infinite electroanalytical techniques in microliter volumes, in preparation. 10 KORYTA,J., PRADAC,J., PRADACOVA,I., AND OSSENDORFOVA,N., Organic oxidation-reduction systems as electrochemical indicators of the monitoring of organs in vivo, Experientia (Basel), Suppl. 18 (1971) 367-374. 11 PRADAC,J., PRADACOVA,J., AND KORYTA,J., Cyclic-voltammetric determination of cysteine in rat organs after intravenous injection, Biochim. biophys. Acta (Amst.), 237 (1971) 450-454. 12 RAJALAKSHMI,R., AND PATEL,A. J., Effect of tranquilizers on the regional distribution of ascorbic acid in the rat brain, J. Neurochem., 15 (1968) 195-199.
209
Voltammetry in brain tissue - a new neurophysiological measurement
PETER T. KISSINGER*, JONATHAN B. HART** AND RALPH N. ADAMS Department of Chemistry, University of Kansas, Lawrence, Kans. 66044 (U.S.A.} (Accepted February 26th, 1973)
A new dimension would be added to neurophysiology if one could implant a sensor into CNS tissue which would respond to concentrations of neurotransmitter substances. Whether or not such a sensor responds in the classical neurological sense (i.e., directly detecting transmitter release upon nerve pathway stimulation) is actually 'frosting on the cake'. Even a direct reading system that would map steady-state concentrations of endogenous neurotransmitters and related small molecular weight materials in living brain tissue would be of great value. At present such data are inaccessible. Sacrifice of an animal followed by conventional analysis of tissue samples provides results which are always uncertain relative to the living system. Our interests in the electrochemistry of catecholamines, ascorbate and other easily oxidized molecules led us to attempt to monitor these substances in CNS tissue. A first step in evolution of an in vivo probe has been accomplished by successfully mating the techniques of electroanalytical chemistry and neurophysiology. The results reported herein are modest and quantitatively uncertain at present, but we believe they are conceptually important and may stimulate others toward practical developments. The standard electrophysiological experiment uses high input impedance devices to measure the potential of a microelectrode with the express desire of drawing little or no current during the measurement. In contrast to these essentially potentiometric measurements, the dynamic electroanalytical techniques monitor finite current. A small (0 ± 1 V) linearly varying potential is applied between a microelectrode and the medium and the resulting current-potential curve or voltammogram is recorded. The voltammogram is characteristic of the nature and concentration of electro-oxidizable or reducible materials close to the electrode surface. Alternatively, a constant potential may be employed and the current monitored as a function of time (i.e., 'chronoamperometry') as the concentration near the surface changes. Reviews of these completely conventional electroanalytical techniques, with special emphasis on solid microelec* Present address: Department of Chemistry, Michigan State University, East Lansing, Mich. 48823, U.S.A. ** Present address: Haight-Asbury Free Medical Clinic, San Francisco, California, U.S.A.
210
SHORT COMMUNICATIONS
trodes and compounds of pharmacological and neurochemical interest are available in the literature~,a, 6. The only familiar application of finite-current electrochemistry to in vivo measurements is the monitoring of oxygen tension with miniaturized Clark-type membrane electrodes5. Recently, Koryta et aL have employed voltammetric measurements at small platinum electrodes to follow cysteine levels in rat kidney and blood10,11. Carbon paste electrodes were constructed from teflon tubing. A press fit stainless steel insert recessed from one end formed a well into which the paste was packed. Electrodes of this very simple design have been manufactured with active-surface dianaeters from about 50 pm to 1.6 mm and details will be presented elsewhere 9. The carbon paste is formulated by mixing oil (Nujol) and graphite (Ultra Microcrystal Grade Graphite Powder UCP-I-M obtained from the Ultra Carbon Corporation) in a ratio of 2 ml oil to 3 g carbon. Conventional Ag/AgC1 (3 M NaC1) reference electrodes in micropipettes were used as were the exposed ends of teflon-coated silver wire (Medwire Corp.) imbedded directly in tissue. Stainless steel, carbon or platinum auxiliary electrodes were equally satisfactory. For in vivo experiments the arrangement depicted in Fig. I proved to be useful, where the chronically implanted stainless steel cannula (typically 18-gauge) served both as a receptacle for the carbon paste electrode and as the auxiliary electrode. Conventional three-electrode instrumentation was constructed from integrated circuit operational amplifiers (details available on request to P.T.K.). Although the Rm
Wm
~
STAINLESSSTEEL
L
TEFLON CARBONPASTE
Fig. 1. Apparatus for voltammetricmeasurementsin brain.
SHORT COMMUNICATIONS
211
CATHODIC (REDUCTION) CURRENT
E vs AglAg CI +0.6 I
,
+0.4 I
,
.~//-~dSCA~ +0.2 I
_
-0.2
_
5 ~
ANODIC
~,.~ (OXIDATION)
~
lo z~
CURRENT
]
-15
Fig. 2. Cyclicvoltammogram recorded from a carbon paste electrode positioned in the caudate nucleus of an anesthetized rat. Apparatus as per Fig. 1.
objectives are different, this circuitry is perfectly analogous to the conventional voltage clamp apparatus in using negative feedback to control a potential difference and measure a current. Fig. 2 illustrates a typical cyclic voltammogram (3 linear cycles of potential from approx. ---0.2 to + 0 . 6 V) recorded with an electrode implanted in the caudate nucleus of an anesthetized Sprague-Dawley rat. Qualitatively similar voltammograms were obtained with electrode placements in cerebral cortex, hippocampus and a variety of other CNS structures. The salient features of this result include the fact that the oxidation reaction does not yield a stable species reducible in the same potential regime (i.e., the voltammogram is said to be 'irreversible'). This is indicated by the lack of reducing current on the reverse half-cycle. Secondly, the dramatic decrease in response from one cycle to the next suggests rapid depletion of the active species in the neighborhood of the electrode. If one waits (either at open circuit or at 0.0 V) for approx. 100 sec before repeating each successive cycle, then the original response is restored. This may be explained by the presence of a thin intercellular solution of low viscosity adjacent to the electrode surface in equilibrium with the surrounding tissue. A small molecule depleted from this region by the electrode will then be refurnished relatively slowly by diffusion from bulk tissue. While we had hoped to detect dopamine (DA) and/or norepinephrine (NE) directly, unfortunately these substances are oxidized at physiological pH at approximately the same potential as ascorbate. It seems probable that the species most likely to account for the voltammogram shown in Fig. 2 is ascorbic acid. This molecule exhibits similar behavior in vitro and is known to be present in CNS at an average level of about 0.3~0.4 mg/g wet tissue 4,12, which is far in excess of that of the catecholamines z,s.
212
SHORT COMMUNICATIONS
However, because of the heterogeneity of the brain, average levels are of questioJ~able utility in this instance. This is particularly true for the catecholamines. F~r example, the DA level in microscopic varicosities in the striatum has been estimated as high as 8000 ng/g 7. The dimensions of these varicosities are, of course, many orders of magnitude below those of the electrodes employed here. Nevertheless, it is encouraging to note that the concentrations of catecholamines encountered even over larger tissue segments are well within the range detectable by modern electrochemical techniquesZ,7, s. It is important to recognize the possible pitfalls of voltammetry in a medium as complex as brain tissue. The heterogeneity of the sample makes the active electrode area uncertain and complicates diffusion of small molecules to the surface. The ability of voltammetry to resolve species with similar redox properties is really quite poor and a single wave may represent several species. Similarly, electroactive material of relatively high concentration (e.g., ascorbic acid) may swamp out the response to minor components (e.g., dopamine) of equal or greater interest. These facts suggest that a completely satisfactory electrode will require some means of controlling geometry and selectivity. Imaginative use of membranes and, perhaps, immobilized enzymes should prov6 helpful ih this regard. Preliminary experiments with electrodes confined behind a dialysis membrane have shown great promise for in vitro experiments, but this approach has been difficult to implement reproducibly in very small electrodes. Ascorbic acid oxidase isolated from squash has been used to rapidly remove ascorbic acid from solutions and it may be possible to make use of this chemistry in an in vivo probe for adrenergic transmitters. Another fundamental concern in developing in vivo probes is the question of disturbing the system with the measuring device. The response of tissue to injury by the probe may be upsetting, at least for short times. These problems are common to any electrode insertion in CNS tissue. The voltammetric currents passed in the experiments described herein should not be any problem since they are in the nanoampere range. This current is considerably below that ordinarily used for physiological stimulation. Despite the concerns described above, the experimental realization that ordinary voltammetry can be carried out in living brain tissue and gives reasonable results is very exciting. Indeed, if development of the present technique succeeds only in providing an in vivo mapping of discrete ascorbate levels, it will be of considerable significance. Our continuing studies are centered on the technological development of smaller electrodes with greater electrochemical selectivity. The support of this work by the National Institutes of Health via Grant 5 R01 NS08740 is gratefully acknowledged. N O T E A D D E D IN P R O O F
We have recently been able to show that this voltammetry technique can be used to reproducibly and quantitatively monitor the concentration-time course of
SHORT COMMUNICATIONS
213
materials injected into ventricle or caudate nucleus tissue. These materials include ascorbate, dopamine, 6-hydroxydopamine (6-OHDA) and, presumably, any electroactive drug system. This style of measurement is of particular significance to neuropharmacological manipulation studies in small animals. Its particular importance to the study of the interaction of 6-OHDA with neural tissue, which at present is still poorly understood, will be presented in the near future (R. McCreery, R. Dreiling and R. N. Adams, unpublished data).
1 ADAM, H. M., The topology of the brain amines - - a review. In G. HOOPER (Ed.), Metabolism of Amines in the Brain, Macmillan, London, 1971, pp. 4-5. 2 ADAMS,R. N., Applications of modern electroanalytical techniques to pharmaceutical chemistry, J. pharm. Sci., 58 (1969) 1171-1181. 3 ADAMS,R. N., MURRILL, E., MCCREERY, R., BLANK,L., AND KAROLCZAK, M., 6-Hydroxydopamine, a new oxidation mechanism, Europ. J. Pharmacol., 17 (1972) 287-292. 4 ALLISON, J. H., AND STEWART, M. A., Quantitative analysis of ascorbic acid in tissues by gasliquid chromatography, Analyt. Biochem., 43 (1971) 401-409. 5 BlCrtER, H. I., AND KNISELY, M. H., Brain tissue reoxygenation time, demonstrated with a new ultamicro oxygen electrode, J. appl. Physiol., 28 (1970) 387-390. 6 HAWLEY,D., TATWAWADI,S. V., PIEKARSKI,S., AND ADAMS,R. N., Electrochemical studies of the oxidation pathways of catecholamines, J. A mer. chem. Soc., 89 (1967) 447-450. 7 HORNYKIEWICZ,O., Dopamine: Its physiology, pharmacology and pathological neurochemistry. In J. H. BIEL AND L. G. ABOOD (Eds.), Biogenic Amines and Physiological Membranes in Drug Therapy, Vol. 5, Part B, Medical Research Series, Marcel Dekker, New York, 1971, p. 192. 8 IVERSEN, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, Cambridge University Press, London, 1967, p. 34. 9 KISSINGER,P. T., DREILING,R., KAROLCZAK,M., ANDADAMS,R. N., Semi-infinite electroanalytical techniques in microliter volumes, in preparation. 10 KORYTA,J., PRADAC,J., PRADACOVA,I., AND OSSENDORFOVA,N., Organic oxidation-reduction systems as electrochemical indicators of the monitoring of organs in vivo, Experientia (Basel), Suppl. 18 (1971) 367-374. 11 PRADAC,J., PRADACOVA,J., AND KORYTA,J., Cyclic-voltammetric determination of cysteine in rat organs after intravenous injection, Biochim. biophys. Acta (Amst.), 237 (1971) 450-454. 12 RAJALAKSHMI,R., AND PATEL,A. J., Effect of tranquilizers on the regional distribution of ascorbic acid in the rat brain, J. Neurochem., 15 (1968) 195-199.