Measurement of the concentration of 5-hydroxytryptamine ejected during iontophoresis using multibarrel carbon fibre microelectrodes

Life Sciences, Vol. 27, pp. 2093-2098 Printed in the U.S.A.

Pergamon Press

MEASUREMENT OF THE CONCENTRATION OF 5-HYDROXYTRYPTAMINE EJECTED DURING IONTOPHORESIS USING MULTIBARREL CARBON FIBRE MICROELECTRODES Z.L. Kruk*, M. Armstronq-James and Jo Millar Department of Pharmacology & Therapeutics*, and Department of Physiology, The London Hospital Medical College, Turner Street, London El 2AD (Received in final form September 24, 1980) Summary Carbon fibre microelectrodes are suitable for electric recording of spike activity in nervous tissues° They can also now be used to measure the concentration of 5-hydroxytryptamine either in bulk solution, or during iontophoreSiSo The electrochemical detection method used allows the time course of appearance and decay of 5hydroxytryptamine during iontophoresis to be closely monitored. A major problem in all experiments employing multibarrel iontophoresis electrode is that the iontophoretically ejected dose is not easily determined° At best, doses can be estimated by reference to the transport number of a similar electrode (1,2), and making assumptions about the volume into which the drug is ejected° A further source of uncertainty is ignorance of the time it takes for a given concentration of ejected drug to accumulate at the receptive site, a parameter which may be called the lag time to reach a particular concentration (3) We report here an electrochemical (4,5) method for accurately measuring the concentration of 5-hydroxytryptamine (5-HT) ejected iontophoretically at the tip of a microelectrode assembly which is also used to record single unit activity in the nervous system° The method is fast enough (15 msec) to follow changes in concentration during iontophoretic ejection, thereby allowing measurement of lag time and other dynamic parameters° Full details of the method are in preparation, the principles are briefly outlined here.

Materials and Methods Three-barrel microelectrodes were prepared as described previously (6) , in which one barrel contained a carbon fibre 7-8 ~m in diameter, protruding 15-20 ~m from the end of the glass and etched to a I ~m tip (7). The second barrel was filled with 2 M NaCI (the reference electrode barrel) and the third with 0 o 0 1 M 5-HT (creatinine sulphate salt) in 0 o 0 1 N HCI. Units were recorded extracellularly in the primary somatosensory cortex of urethane-anaesthetised rats° For recording units, the carbon fibre electrode was switched into an FET voltage follower headstage and the preparation grounded in the normal way° Carbon fibre microelectrodes when used for single unit recordln~ have low noise and therefore produce high signal-to-noise ratio recording (6,81o Concentration of 5-HT at the electrode tip was measured both in vitro and in vivo. For in vitro assay, the microelectrode array was lowered into a beaker of 0°9% NaCl, along with a chlorided silver wire (the auxiliary electrode). The carbon fibre electrode was switched from the voltage follower headstage to a "virtual ground" current-to-voltage convertor headstage.

0024-3205/80/482093-06502.00/0 Copyright

(c) 1980 Pergamon Press Ltd.

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A + 0.7 v o l t I00 Hz sawtooth w a v e f o r m from a function generator was fed into an analog gate so that only 1½ cycles w e r e o u t p u t for every fifty input. Thus, a t h r e e - p e a k e d t r i a n g u l a r w a v e f o r m lasting 15 m s e c was p r o d u c e d (Fig° IA) every 500 mSeCo The tip of the m i c r o electrode assembly was forced to follow this voltage p a t t e r n by connecting the r e f e r e n c e b a r r e l of the m i c r o e l e c t r o d e to a voltage follower c o n n e c t e d in turn to a negative feedback circuit with the a u x i l i a r y electrode as the o u t p u t to the saline° During this v o l t a g e p a t t e r n at the tip, a current flowed through the carbon fibre in accordance w i t h the e l e c t r i c a l impedance of the system, w h i c h has an e q u i v a l e n t circuit of a resistor in series w i t h a "leaky" capacitor (8).

Cathodic (reducing) 0.0v or -400mV Anodic (oxJdising)

B Oxidation current

Reduction current

FIG.

I

Fig. IA shows the v o l t a g e w a v e f o r m p r o d u c e d at the tip of the carbon fibre by the voltage clamp circuitry. P e a k - t o - p e a k v o l t a g e was 1.4 volts, total d u r a t i o n of w a v e f o r m 15 m s e c (calibration bar is 5 msec). Fig° IB shows on the same time scale the c u r r e n t flow through the carbon m i c r o e l e c t r o d e caused by the voltage w a v e f o r m shown in Fig. IA. Two s u p e r i m p o s e d traces are s h o w n , o n e obtained in v i t r o (in saline) and one (marked by arrowheads) o b t a i n e d in vivo (in the rat cerebral cortex). Results

and D i s c u s s i o n s

D i f f e r e n t m i c r o e l e c t r o d e s g e n e r a t e d slightly d i f f e r e n t current waveforms, but they all were similar to those shown in Fig° lB. Two current "sweeps" from one m i c r o e l e c t r o d e are shown in Fig. IB, one p r o d u c e d w i t h the m i c r o e l e c t r o d e tip

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in saline, the other w i t h the m i c r o e l e c t r o d e tip 1,000 ~m b e l o w the surface in the s o m a t o s e n s o r y cortex of an a n a e s t h e t i s e d rat. For the latter current, the a u x i l i a r y e l e c t r o d e was b u r i e d in the r e t r a c t e d neck muscle. It can be seen that the in v ~ t r o and in v i v o sweeps were p r a c t i c a l l y identical° When 5-HT was added to the solution in vitro, e l e c t r o - o x i d a t i o n of the amine o c c u r r e d w h e n the carbon fibre was at a p o s i t i v e p o t e n t i a l (anodic). The liberated electrons p a s s e d into the carbon fibre and p r o d u c e d an i n c r e a s e d anodal current flow over that found in saline. The 5-HT o x i d a t i o n p r o d u c t s (s) were reduced d u r i n g the s u b s e q u e n t cathodal cycle of the carbon and p r o d u c e d a c o r r e s p o n d i n g cathodal current increment. By using the fast ramp p a t t e r n shown in Fig. IA, there is little net c o n s u m p t i o n of 5-HT and little or no p o i s o n i n g of the electrode surface with i n s o l u b l e o x i d a t i o n products.

B

' .5 m S E C

lo-5

lo -4

MOLAR C O N C N

lo-3 5-HT

D

FIG.

2

Fig~ 2A shows a set of nine s u p e r i m p o s e d current sweeps p r o d u c e d in saline and in a set of saline solutions containing i n c r e a s i n g concentrations of 5-HT from 10 -5 M to 10 -3 M. The greater the concentration, the larger the i n c r e m e n t p r o d u c e d by o x i d a t i o n and r e d u c t i o n of the 5-HT. The d i f f e r e n c e b e t w e e n the saline current and the saline plus 5-HT o x i d a t i o n current was m e a s u r e d at the p o i n t indicated by the filled triangle. Fig. 2B shows a c a l i b r a t i o n curve d e r i v e d from Fig. 2A of incremental c u r r e n t (deflection at p o i n t m a r k e d by triangle) v e r s u s m o l a r c o n c e n t r a t i o n of 5-HT. Fig. 2C shows a set of p o l a r o g r a p h i c c u r r e n t sweeps from the same m i c r o e l e c t r o d e as in Fig° 2A, in saline, w i t h a series of steady i o n t o p h o r e t i c currents p a s s e d through a 5 - H T - c o n t a i n i n g b a r r e l in the m i c r o e l e c t r o d e . I n c r e a s i n g the i o n t o p h o r e t i c current i n c r e a s e d the o x i d a t i o n p o l a r o g r a p h i c current, but in an u n e v e n fashion. Ten sweeps are shown superimposed, as (from smallest trace to largest) i0 n a n o a m p e r e s (nA) r e t a i n current, 0 nA (passive leak state) then i0, 20, 30, 40, 60, 80, 100 and 200 nA eject current.

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Fig° 2D shows a set of polarographic current sweeps taken in vivo using the same set of iontophoretic currents as in Fig° 2Co The signals (measured after one minute of steady iontophoretic current in each case) were considerably smaller in vivo than in vitro, possibly reflecting active uptake in vivo. The increment in current flow produced by 5-HT was found to be related to the concentration of 5-HT present at the tip of the electrode. Fig° 2A shows a series of current sweeps in a set of saline solutions containing different concentrations of 5-HT. The greater the concentration of 5-HT, the larger the current sweeps obtained° Fig° 2B shows a calibration curve constructed from Fig. 2A by measuring the peak oxidation currents as a function of 5-HT concentrationo If the electrode was now transferred to a fresh beaker of saline, a series of current traces could be obtained during iontophoretic ejection of 5-HTo This is shown in Fig. 2C. Under steady state conditions the concentration of 5-HT at the tip was found to be dependent upon the iontophoretic current. Thus, by using the curve obtained in Fig. 2B, the concentration of 5-HT at the tip of the electrode caused by iontophoresis could be estimated° When the same procedure was repeated in vivo, a similar set of current sweeps was obtained (Fig. 2D). It should be noted that in preliminary experiments with 5-HT, the microelectrode tip was clamped at ground potential between current sweeps. This procedure was adequate to prevent poisoning of the electrodes during experiments with dopamine or noradrenaline (unpublished observations)° However, with 5-HT, a progressive reduction in size of the reducing current increment was noted. In later experiments, therefore, the tip potential was clamped at -400 mV (reducing) between sweeps. This greatly improved the reliability and reproducibility of the results.

o

5 TIME

-

lO

MINUTES

FIG. 3 Time course of 5-HT concentration changes at the microelectrode caused Dy iontophoretic ejection. The signal was measured in

tip

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arbitrary units as the difference at the oxidation peak between the polarographic current with 20 nA retain and the current during iontophoretic ejection° The curve indicated by square markers was obtained with 50nA ejection current, those with circles and triangles were from 25 nA and 12.5 nA respectively. The period of ejection current is indicated by the horizontal bar° Using carbon fibre microelectrodes, the lag time for the ejection of 5-HT was investigated° After a fixed duration (5 minutes) of 20 nanoamperes (hA) retain current, a variable level of eject current was switched into the 5-HT iontophoresis barrel of a three-barrel carbon fibre microelectrode immersed in saline. It was found, firstly, that for many microelectrodes very little 5-HT was ejected for the first 10 seconds or so after current switch-on. This was especially noticeable when small eject currents (< 40 nA) were used+ Secondly, the concentration of 5-HT at the tip of the microelectrode had not reached a steady level even after one minute of continuous iontophoretic current in the majority of microelectrodes° This result is illustrated in Fig. 3, which shows the time taken to reach a steady 5-HT concentration at the microelectrode tip using three different levels (12o5 hA, 25 nA, and 50 nA) of ejection current in a typical microelectrodeo The duration of 'retain' current prior to the ejection current switch-on also had a significant effect on the lag time. The three sets of data in Fig. 3 were all taken after a fixed period of 5 minutes retain current of 20 nAo

100 +

Z

_o

•

t/)

/

/ /

~50,

+e

025

0.5

1"0 TIME

-

FIG.

2:0

5"0

MINUTES

4

Effect of retain current duration on the time course of 5-HT concentration rise at the microelectrodeo The curves were all obtained using a fixed ejection current of 25 nA. Each ejection current was preceded by a different duration of 20 nA retain current° The top curve marked by open triangles was obtained after 0.5 min retain current, and the lower curves from longer durations as follows; filled triangles: one minute~ open squares: 2 minutes; filled squares: 4 minutes; open circles: 8 minutes; filled circles: 16 minutes.

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Using the same microelectrode, the lag time was analysed using a fixed ejection current of 25 nA, but preceded by a variable duration of 20 nA retain current. Fig° 4 shows the curves resulting from (from top to bottom) 0.5, I, 2, 4, 8 and 16 minutes retain current duration preceding the ejection. For 16 minutes of retain current, the concentration o~ 5-HT had only reached about 60% maximum even after one minute of ejection° These results provide direct support for a recent theoretical analysis of temporal factors in iontophoresis (Ii) . In summary, the technique of high-speed electrochemical detection enables the precise concentration at the microelectrode tip of iontophoretically ejected 5-HT to be monitored° The same microelectrode assembly can be used for lownoise single-unit recording at the ejection site° Preliminary observations on the lag time of iontophoretically ejected material suggest that it may take several minutes for steady levels of concentration to be built up at the microelectrode tipo

REFERENCES Io 2° 3. 4. 5o 6° 7. 8° 9° i0o

HOFFER, B.J., NEFF, No and SIGGINS, G~R., Jo Neuropharmacol. I_~0, 175-180, (1971) BRADLEY, P.B. and CANDY, JoM., Brito J. Pharmacolo 4_~0, 194-201, (1970) BLOOM, F.E., Life Sci. 14, 1819-1834, (1975) McCREERY, RoL., DREILLING, R. add ADAMS, R.N., Brain Res. 73, 23-33, 1974) BOCKRIS, Jo O'Mo, and DRAZIC, D., Electro-Chemical Science. Taylor and Francis, London. (1972) ARMSTRONG-JAMES, M. and MILLAR, Jo, Jo NeuroSCio Methods. ~, 279-287, (1979) ARMSTRONG-JAMES, M. and MILLAR, J., J. Physiolo (An press) 1980o ARMSTRONG-JAMES, Mo, FOX, K. and MILLAR, Jo, J. NeuroScio Methods (in press) (1980) FOX, K., ARMSTRONG-JAMES, M. and MILLAR, J., Jo Neurosci. Methods° (In press) (1980) PURVES, ROD., Jo Neurosci. Methods° ~, 165,178, (1979) o