topic/ in chemic~lin~trument~tion
edited by
FRANK A. SETTLE, JR. v i ~Lexington. i n i a M l i tVA a r y24450 l~titule
Approaches to Voltammetric and Chromatographic Monitoring of Neurochemicals In Vivo Alice J. Cunningham' Agnes Scott College, Decatur, GA 30030
Joseph B. Justice, Jr. Emory University, Atlanta, GA 30322
During the past 15 years there has been considerable oroeress .. in the develooment of m < t h d i for mmitonna neuruchem~calsin w v o Priur to the I d 7 report by Kissing*,. Hart. and .\dami. I 1 ) pertaining to wltatnmetry in brain tissue, there had been some pioneering in vivo work by Kortya's group (2, 3). Using cyclic voltammetry they measured cysteine in kidneys and blood. Although the electrochemical techniques may be the same as those used for classical in wrnr elecrn,nnal)ri~.alexperrmenm, the implantation of elertn,den into livmg tissue imposes a number of requirements for alteration of the methodology. These requirements are, of course, dictated by the nature of the tissue and the dynamics of the processes being monitored. The same holds true for chromatographic analyses of brain fluids, or any other body fluids. Sampling and transfer become the major factors of consideration in this latter case. In this article we shall discuss some of the unique prohlems confronting the chemists who do in vivo voltammetry or chromatographic sampling of the extracellular fluid (ECF) of the brain. Many of the developments have evolved from mod common sense aoolicatmn or analgtlcnl prinr~ples,hut the implementerim nlsu requwes considsmhleskill in refinement* t r f methuds and rhorutrgh knowledge of the apparatus and experimental approaches of many areas of neuroseience. One of the first problems is the miniaturization of the apparatus. Implantation is not difficult; conventional stereotaxic procedures are used for locating the various regions of the brain (4) and routine surgical methods are utilized. The size of the implanted devices, however, is a major consideration. The electrode or sampling cannula must be small enough to sample a very localized region with minimum damage during imolantation. In eeneral. the size limits are
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minimal damage considerations. At the lower limit one has to consider the possibility of sampling from a cell interior as opposed to sampling from the extracellular fluid. When one considers the sizes of cells (5) in the A34
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brain (10-20-pm diameters for the neuronal cell bodies and less for extraneuronal processes) it becomes immediately obvious that microelectrodes with diameters much Less than 10 r m are desirable-the smaller the better. The reality of actually measuring events in the synaptic clefts is remote, however, since they have a width of about 0.02L 0.4 pm, depending on the type of synapse. The experimental compromise imposed by current technology has resulted in routine usage of bl0-rm-diameter electrodes. The chromatographic sampling cannulae commanly in use have diameters of about 200300 pm. Thus, one samples from the extracellular fluid of the brain by insertinga relatively large device into the intricate network of varied cells, axons, dendrites, etc.
ters of about 10-40 r m and working electrade surface diameters of 5-20 r m . Carbon paste, carbon epoxy, and carbon fibers are the most common electrodes in most laboratories. In fabricating these electrodes a micropipet is pulled to the desired capillary diameter using a capillary puller. The working electrode material is packed or inserted into the capillary opening. Electrical cont a c t is made via conventional means throughthe pipet barrel. Details for construction of single and multibarrel electrodes have been reported (10,lI). Each of these electrode materials exhibits a different selectivity for the endogenous materials in the extracellular fluid of the brain (see refs 6-9 for a mare comolete discuislunl. Efforts to retine selectivities have been neressitatrd by the ut,iquitrms presence uf aicarhir aeid and the clme $,alumfor The effects of electrode surface alterthe redox potentials of the various neuroations upon contact with brain tissue and transmitters and their metabolites. Figure 1 the problems of interferences due t o the illustrates the problems of overlap of the myriad endogenous substances must ultioxidation potentials for some of the more mately be identified and accounted for. This important electroaetive species and the is not easy, but the reproducibility of results wide concentration variation. Modification is actually amazing considering the comof the electrode surfaces shifts several of plexity of the matrix in which one works. these observed potentials. For example, a Several reviews with details of voltammetric carbon fiber, particularly when pretreated and chromatographic methodologies are electrochemically, is more selective for the available (6-9). neun,rrnnsmittcr dopamme than ior its metahcrlic DOI'AC 13.4-dihydroxyphenylVoltammetry In Wvo aretir e c d ) 8.r ior airurbiu arid. The carbon Lrt us n m l d e r the clectrudrs fur in v ~ w paste electrode may be altered for dopamine selectivity by introducing stearic aeid into vultammetr~rmeasurements. 'l'hc reference the paste formulation. The application of a and suxil:ary electruder it,, thrcc-electrude Nafion (sulfonated fluorocarbon) film to electrochemical cell arrangements are fairly carbon epoxy electrodes also makes them simple. The auxiliary electrode can be as selective for positively charged species such simple as a stainless steel wire or a cortical as dopamine, other catecholamines, and sescrew used during surgery. The reference rotonin (5-HT). Reference 9 should be conelectrodes are commonly micro AgIAgCI sulted for a mare complete discussion of electrodes. This is a convenient reference electrode modifications currently in use. since the extracellular fluid is about 0.15 M Potentiostats used far in vivo voltamin Cl-. Placement of the reference and auxmetry are basically quite conventional deiliary electrodes in the brain is not critical. signs (12, 131, except for enhanced current The extracellular fluid is a highly conductsensitivity. The instrument must be capable ing medium and the currents passed a t the microelectrodes are in the nano- or pieoamp ranee. Thus. iR droo is neelieible. F w w r k l n r . electrodca most researchers ui* one ot' three t)pes uf a r b o n clc~lrudes (Continued on page A36) tdl,ricated t u have enratemmr bod! diawu-
(Concentration Ranges) (Resting Levels)
of generating various waveforms for the different eleetroanalytieal methods, and it must sense very small currents with good time response. Systems currently in use are microprocessor-controlled since the data must be stored and there must be computer control of data manipulation such as suhtraction of background and averaging. Waveforms obtained from a function generator or generated by computer software and processed through the digital-to-analog converter of the microprocessor interface are used for input t o the potentiostat. A variety of electrochemical techniques have been applied in the study of brain chemistry. While ehronoamperometry has been one of the most widely used methods for time-dependent processes, cyclic voltammetry, pulse voltammetry, and semi-differential voltammetry have been utilized in numerous studies, particularly where qualitative information is required. There are some particular considerations in instrument desien that should he menricmed. T h e extremely lou roncentraticms (nnm,mdnr to micnmdar), rwpled wrth the very small microelectrode areas, produce currents that are in the range of pieo to nanoamps. This requires an instrument of extremely low noise level-thus high quality operational amplifiers, thorough grounding, and extensive shielding. For example, connections to the working electrode are usually made directly t o the pin of the circuit board, thereby eliminating excessive counections. A Faraday cage is an essential component when minimizing noise; most researchers put the animal and all potentiostat components except the power supply in the cage. Gerhardt and Adams have described a n instrument t h a t is hatter" powered 1 1 4 1 This approach reduces c m sidcrably i h r problem o i power supply noise. In choosing operational amplifiers, the characteristics of greatest concern are: 1) very short rise time, 2) high . input impedan& and 3) low input hias current andlow input noise. In this laboratory the Analog Devices 515 and RCA 3140 amolifiers have been adequate ftx most spplicatmns. These haw 1 0 . R input rmpedances, very high slew rates, and low input noise characteristics. Figure 2 is a schematic of one patentiostat design used in this laboratory. Presently a Keithley Model 427 Current Amplifier is being used in conjunction with a 12-bit Data Translation 2801A interface to a Zenith 158 microcomouter. Multi-electmde potentroktots nuu in use in most laboralurirs e r e a ~ m h 1*, r the Hlakely nnd 1)uvarney 0 5 ) design in which each derrrude has its own two-stage input amplifier arrangement. This arrangement, as opposed to one in which there is a multiplexer, eontributes even further to noise reduction in the system. The Armstrong-James group in England has described a fast cyclic voltammetry system that utilizes a function generator and waveform gate for 100 Hz cyclic voltammetry (16). Output is directed to a digital transient oscilloscope. Complete cyclic vol-
Figdm 1. Comparison of ox dataonpmentiala and concentration ranges tor several endogenous suostancss 01 extmcell~larflu d. Abbreviatims: DA. dopam ns; DOPAC. 3.4dihydroxyphenylacet1~ =ad:AA. a 6 C O m C ac d; rlVA, hamovanillic acia. 5-HI, serotonin (5-hydroxqtryplamine):6HIAA. 5-hydroxyindolsacstic acid. Superscripts: carbon paste; carbon fiber, untreated: carbon-epoxy, untreated
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tammetric scans in the potential region of interest are possible in 15 ms or less with this system. Variations of this design should be invaluable in following the transient responses for short-lived species involved in neurotransmission.
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Journal of Chemical Education
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Figure 2. Potentiostat and eurrem amplifier. All resistors are 118 W. 1 % . OAl is a Ti072 dual op amp. OA2 and OA3 are RCA CA3140S wideband ap amps. The 12-bit AdaiabIAi13 imerface is available from Interactive Microware. inc.. State College. PA. Assembly language routines are used to achieve 0.05 msl~ampie.
Sampling and Chromatography of Braln Fluld Figure 3 shows the schematic for a typical cannula used for chromatographic sampling of brain dialysate and an entire assembly for on-line sampling and injection. First introduced by Ungerstedt's group, the technique-push-pull perfusion of brain dialysates-has recently been the subject of a complete review (17). The basal levels of many neurochemicals are in the nanomolar t o micromolar range. The experimental trick is t o "select", with minimum perturbation of the brain microenvironment, the desired components through t h e dialysis membrane and transfer them to the head of the column with minimum dilution and t i m e delay. The dialysis membrane prefilters the sample for chromatographic analysis and minimizes tissue damage by maintaining fluid flow and any turbulence inside the dialysis tube. In 1983 model studies for brain dialysis and of high-performance liquid chromatographic analysis of the dialysate were reported (18). The interrelated effects of eannula length, flow rate, and concentration were examined. A recent report from this laboratory (19) contains details about sampling considerations for microbore, or small bore, chromatographic analyses (LCEC) of brain dialysates. In this study i t was shown that under microbore conditions there are practical tradeoffs when considering flow rate, sample size, and detection limits. Using the microbore approach, however, sensi(Continued on page A39)
An~malChamber
Recorder
Figure 3. (a) below-typical elenrode assembly fw in vivo voltammeby. Right-typical dialysis cannula for on-line chromatography sampling. Length Is adjustable. Fused silica tubing inside dialysis cannula goes directly into injection pan. The dialysis tubing has a 5000 MW cutoff. Reprinted with permission from American Laboratory (1986. 18(10), 33). International Scientific Communications. Inc.. Fairfield. CT). (b) (top) Schematic of monitoring system for chromatographic analysis of dialyzed perfusate. Reference 19 should be consulted for experimental details.
tivity and sampling times are enhanced significantly. The report cited above (19) describes a system thathas adetection limit of 300 fg of dopamine using a perfusion flow rate of Figure 4. Chromatogram showing dopamine peaks before (- - - - ) and aner (-) adminishation of amphetamine ( I mglkg, i.p.) to anesthetized rat. A 5pm. C18 microbore (1.0 mm X 10 cm) column was used with 6min sampling intervals.
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February 1987
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rat. The actual choice of operating parameters far chromatographic analysis depends basically on the nature of the experiment. If one is studying the longer term effects of drugs or performing experiments an multiple-component systems involving several transmitters and their metabolites, a slow sampling rate and thorough separation of components is desirable. In contrast, when examining the relationship of a single neurotransmitter t o behavior, a high sampling rate and fast chromatographic separation is more desirable.
Appilcatlons While much of the literature in voltammetric and chromatographic monitoring of neurochemicals has dealt primarily with methods development, a review of applications to date reveals the extensive possibilities for understanding brain function. As early as 1979 Curzan e t al. (20) correlated voltammetric results with stressful manipulations via tail pinch in rats. Several other groups have examined the relationships betwe& voltammetric responses and movement @I),feeding patterns (22), emotional excitation (23). and hormonal variations resulting fromsi&ulatedsuekling (24,25).Michael e t al. (26) have determined kinetics of the metabolic conversion of released dapamine to 3,4-dihydroxyphenylaceticacid, DOPAC. Also, Wightman's group (27) has determined the uptake rate of released dopamine into cells of the brain's mieroenvironment. Fieure 5 illustrates twical results from the !;udy by Michael e; ;I. (2fil. T h c meni u n m t m i were nmdr in the srrintum of a n anesrhr~izedrat. Aiter allowing the inrt~al ampenmetric response to decay to a steady wlw 0.5 nnmp, an electrical strmulstion t , i the medlsl forebrain hundlr was applied. There was an immediate short-term ;&perometrie response, probably due to dopamine (DA), then a slower rise in signal due to theDA metabolite, DOPAC (curve A). After 60 min pargyline, a drug which inhibits conversion of DA to DOPAC, was administered (100 mghg). A new baseline of 0.29 namp was established, then the electrical stimulation was repeated. As seen in curve B, the signal due to DOPAC was not observed because the DA-to-DOPAC conversion had been blocked by the drug action. From data such as this, the authors reported the kinetic information for appearance and clearance of DOPAC in the extracellular fluid of the brain. This general experimental approach may he used in a variety of studies designed to investigate drug effects which should correlate with voltammetric and chrornatographic results.
Summary From this discussion, one can see that the extensions of voltammetry and ehrornatography to in vivo applications have neeessi-
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Journal of Chemical Education
TIME IN MINUTES. Figure 5. In vivo chronoamperomehicrespanseof electrade implanted In the striatvmofan anesthetized rat. Curve A is beforeadminisbation of pargyline. Curve B results after administration of pargyline (100 mglkg). See text for discussion. Re~rintedwith oermission from American Labwatory (1988, 18(10).33). International Scientific Communications.Inc.. Fairfield. CT
tsted the development of several new measurement approaches far the analytical chemist. Primary emphasis has been on design of smaller sampling devices, alterations of electrode surfaces t o improve selectivity for the endogenous substances of extracellular fluid, careful attention to the factor of time resolution for widely varying time domains, and exceptional enhancement of SIN characteristics. As neuroscientists attempt to elucidate further the intrrrate i n teractions ofcomplex processes mvolved in neunrtransmissiun. it is predictable that even mare ingenious experimental techniques will be required. One can project t h a t significant advances will be forthcoming in thk use of voltammetric and chromatoeraohie methods for monitorina neurochemicals and correlating changes with normal neurulmrtiun, pharmacological effects, nervous svscpm dysfunction, and behavior.
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1111 Gerhardt,G.A.;Adamn,R. N.An.1. Chem. 1982.54. 1888.
(15) B1akely.R. D.: Duvsmey. R. C. BroinRos. Bull. 1983. 10.315. 1161 Armstrong-James,M.; Fox, K.: Kruk. 2. L.: Millar, J. J. Neurosei. Methods 1981.4.385. 1171 See ref 6. Chap 4. (181 Johnson, R. D.; Justice, J. B. Blain Re$. Bull. 1983, 10,567. 1191 Wapel. S. A.; Church, W. H.;Justiee. J. B.,Jr.Awl. Chem 1986 (in press). 120) Cu.ron.C.:Hufso",P.H.:K"ott,P.J.R~~J.Phhhh0. rol. 1979,66,127P. 1211 Yamamoto. B. K ; Lane, R. F.;Freed, C. R. Life sci. 1982.30.2155. 1221 Satamone.J. D.:Lindsay, W. S.: Nei1l.D. B.: JuLies, .I. B. 1985, manuscript in prep. 128) Lindsay.W. S.: Horndon, J. G.. Jr.; Blakely, R. D.; Justice.. J. B... Jr.:. Ncill. D. B. Brain Re8. 1981. 220, 891. (21) P I O U ~ P. ~ , M.: Neil1.J. D. Endocrinology 1982, 110, ~
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691.
(251 Plotsky, P. M.: DeGreef, W. J.: Neill,J. D. Broin Reg 1982,250,251, 1261 Michael. A. C.: Justice, J. 8.. Jr.; Neill, D. B. Neurorci. Lett. 1985.56.365. 1271 Ewing. A. G.; Wightman. R.M. J. Neumchzm. 1984. 43.670.
1 Visiting scholar. D e p a m n t of Chemistry, Emory University, Atlama, GA. 1984-88.
Literature Clted (11 ~irninger,P.T.;Harf,J.B.:Adams,R.N.Bmin Re. 1973,55,209. 121 Konya. J.: Pradac. J.: Pradaeovs, J.; Cmendorfova. N. Experisnfia Suppl. 197I,I8,367. (3) Pradac, J.: Pradamvs. J.: Kortya, J. Riochim. Biophys. Ado iAmal.1 1971,237,450. (11 Pellegcino, L. J.; Cushman,A. J. "A Stcreotaxic Atlas of the Rat Brain": Appleton-Century-CmfU:New York. ,967. (5) Psrik, P.; Pasik, T.; DiFiglia, M. In "The Nerratriaturn": Diva
