Electroencephalography and Clinical Neurophysiology, 1 9 7 8 , 4 4 : 3 9 8 - - 4 0 2
398
© E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers, Ltd. Technical
contribution
A GATED, HIGH MONITORING
VOLTAGE
IONTOPHORESIS
SYSTEM
WITH
ACCURATE
CURRENT
B.M. S M I T H a n d B.J. H O F F E R *
Section on Technical Development, National Institu te o f Mental Health and National Institute o f Neurological and Communicative Disorders and Stroke, Bethesda, Md. 20014 and Laboratory o f Neuropharmacology, National Institute o f Mental Health, St. Elizabeth's Hospital, Washington, D.C. 20032 (U.S.A.) ( A c c e p t e d for p u b l i c a t i o n : J u n e 15, 1 9 7 7 )
T h e local a p p l i c a t i o n o f drugs f r o m m u l t i b a r r e l m i c r o p i p e t t e s b y m i c r o i o n t o p h o r e s i s has p r o v e n t o be o f great i m p o r t a n c e in e l u c i d a t i n g drug a c t i o n s o n specific areas of t h e n e r v o u s s y s t e m . By c i r c u m v e n t ing m a n y o f t h e m e c h a n i c a l a n d e n z y m a t i c barriers p r e s e n t in t h e b r a i n a n d m i n i m i z i n g r e m o t e or indir e c t d r u g a c t i o n , this t e c h n i q u e has allowed precise c h a r a c t e r i z a t i o n of a n u m b e r o f p u t a t i v e n e u r o t r a n s m i t t e r s ( H o c k m a n a n d Bieger 1 9 7 6 ) , as well as pharm a c o l o g i c a l agents w h i c h i n t e r a c t w i t h discrete classes o f synapses. As w i t h m a n y i n n o v a t i o n s , it h a s b e c o m e a p p a r e n t t h a t m i c r o i o n t o p h o r e s i s possesses its o w n p a r t i c u l a r set of t e c h n i c a l l i m i t a t i o n s a n d pot e n t i a l artifacts. A m o n g t h e m o s t i m p o r t a n t are: (1) failure of t h e p i p e t t e to release drug ( H o f f e r e t al. 1 9 7 1 a ) , (2) p o l a r i z a t i o n of the n e u r o n f r o m i o n t o p h o r e t i c c u r r e n t ( H o f f e r et al. 1 9 7 1 b ) a n d ( 3 ) a l t e r a t i o n s in a m o u n t of drug released due to c h a n g e s in t h e t i m e b e t w e e n d r u g e j e c t i o n s ( B r a d s h a w e t al. 1 9 7 3 ; F r e e d m a n e t al. 1 9 7 5 ) . This p a p e r describes an i o n t o p h o r e s i s s y s t e m t h a t p r o d u c e s a c c u r a t e drug e j e c t i o n a n d allows p r o p e r i n t e r p r e t a t i o n o f drug r e s p o n s e s b y p r o v i d i n g m o n i t o r s o f critical i o n t o p h o r e t i c p a r a m e t e r s . T h e s e novel f e a t u r e s are incorp o r a t e d i n t o an i n s t r u m e n t t h a t has t h e same general c o n f i g u r a t i o n as t h a t d e s c r i b e d b y Geller a n d Woodw a r d ( 1 9 7 2 ) : t h r e e c u r r e n t s o u r c e s p e r m i t applicat i o n o f t h r e e s e p a r a t e drugs while a f o u r t h b a l a n c e c h a n n e l m i n i m i z e s n e t c u r r e n t flow to g r o u n d . A c c u r a t e drug a p p l i c a t i o n t h r o u g h the m u l t i b a r r e l m i c r o p i p e t t e , w h o s e resistances can be several h u n d r e d m e g o h m s a n d o f t e n s h o w m a r k e d variability w i t h time a n d c u r r e n t passage, is p r o d u c e d by solids t a t e v o l t a g e - c o n t r o l l e d c u r r e n t sources w i t h high outp u t i m p e d a n c e a n d high voltage c o m p l i a n c e . In addition, t h e g e n e r a t i o n of the drug c u r r e n t for each o f
* P r e s e n t address: Dept. of P h a r m a c o l o g y , University of C o l o r a d o S c h o o l of Medicine, Denver, Colo. 8 0 2 2 0 , U.S.A.
t h e t h r e e p u m p i n g c h a n n e l s m a y be s y n c h r o n o u s l y c o n t r o l l e d b y a digital gate signal f r o m an e x t e r n a l t i m i n g device. This facilitates b o t h regular, periodic drug e j e c t i o n a n d c o m p u t e r analysis o f n e u r o n a l r e s p o n s e s ( F r e e d m a n e t al. 1975). T h e critical p a r a m e t e r of i n t e r e s t in i o n t o p h o r e s i s is t h e drug c u r r e n t actually b e i n g delivered t h r o u g h t h e m i c r o p i p e t t e . With a v o l t a g e - c o n t r o l l e d c u r r e n t s o u r c e p r o v i d i n g t h e c u r r e n t , the c o n t r o l voltage is generally used as t h e c u r r e n t m o n i t o r . However, with t h e n e e d to pass relatively large c u r r e n t s t h r o u g h high resistance p i p e t t e s , t h e p r o d u c t of c u r r e n t a n d resist a n c e c a n e x c e e d the voltage c o m p l i a n c e of the curr e n t source. W h e n this h a p p e n s , t h e linearity b e t w e e n c u r r e n t a n d c o n t r o l voltage ceases a n d the c o n t r o l voltage gives a false i n d i c a t i o n of drug c u r r e n t . T h e c u r r e n t s o u r c e design p r e s e n t e d here p r o v i d e s a m e a n s o f g e n e r a t i n g a c u r r e n t m o n i t o r t h a t gives a true reading even w h e n the voltage c o m p l i a n c e o f t h e c u r r e n t s o u r c e is e x c e e d e d . This t y p e of c u r r e n t m o n i t o r i n g r e d u c e s false negative findings of drug efficacy a n d p e r m i t s the inclusion of an u n b a l a n c e c u r r e n t m o n i t o r . T h e u n b a l a n c e m o n i t o r i n d i c a t e s w h e n the difference b e t w e e n the t o t a l drug c u r r e n t f r o m t h e t h r e e p u m p i n g barrels a n d t h e c u r r e n t f r o m the b a l a n c e barrel e x c e e d s a preset value. A n y s i g n i f i c a n t u n b a l a n c e generally indicates t h a t the voltage c o m p l i a n c e of o n e of t h e c u r r e n t s o u r c e s has b e e n r e a c h e d following a partial blockage of t h e c o r r e s p o n d i n g barrel of t h e m i c r o p i p e t t e . Moreover, this u n b a l a n c e alerts the e x p e r i m e n t e r t h a t a n y o b s e r v e d n e u r o n a l r e s p o n s e s m a y be due to electronic effects of the iontophoretic current, rather t h a n to the drug. A typical e x p e r i m e n t a l a p p l i c a t i o n is s h o w n in Fig. 1. I o n t o p h o r e s i s of 55 n A o f 13-endorphine ( E 5 5 ) , a 31 a m i n o acid p o l y p e p t i d e believed to be an endogen o u s o p i a t e ( T e s c h e m a c h e r e t al. 1 9 7 5 ) , p r o d u c e d a r e p r o d u c i b l e i n h i b i t i o n of firing of this rat c a u d a t e n e u r o n . T h e effects of 13-endorphine are a n t a g o n i z e d by i o n t o p h o r e s i s of 10 n A of the o p i a t e a n t a g o n i s t ,
IONTOPttORESIS SYSTEM
399
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3o ";";i Fig. 1. Effects of iontophoresis ~-endorphine and naloxone (N) on spontaneous activity of a single caudate neuron in rat. The ratemeter record is continuous in time. The magnitude of the ejection current in nanoamperes is indicated by the numbers after each drug and the duration of ejection, by the underlying bars. Note that ~-endorphine elicits a powerful depression of spontaneous discharge rate, and this response is reversibly antagonized by naloxone. Similar interactions were seen in all six caudate neurons tested. Initial drug barrel resistances for these substances ranged from 75 to 300 M ~ .
naloxone (N10). This experiment illustrates a number of the advantages of the circuitry described here. First, utilization of the gate input to eject ~-endorphine at regular intervals produces uniform control inhibitions. Second, the high compliance of the pump plays an important role, no doubt, in obtaining prompt neuronal responses to this large 31 amino acid polypeptide with high drug barrel resistance. Finally, unbalance, in this experiment, was less than 10 nA, minimizing any possibility of electrotonic artifacts.
Circuit description The principal circuit components of the system are divided into 4 sections in Fig. 2: (A) control voltage amplifier; (B) current source and high-voltage regulators; (C) current monitor, and (D) unbalance current monitor. Amplifier A1 combines 3 input voltages to generate the negative control voltage --V c for the current source. The 3 inputs are (1) a DC voltage to generate a drug-holding current, (2) a second DC voltage for drug-pumping current which may be applied continuously by manual switch or pulsed on by an external
digital gate input and (3) an analog input used in place of (2) when a pulse generator with multiple calibrated outputs is available. The value of the DC 'holding' input is set by the 1K, 10-turn potentiometer, the 5 or 50 nA/turn range switch, and the polarity switch. The second DC voltage is generated in exactly the same manner but must pass through the solidstate analog switch VS. VS is controlled by the optical isolator OPI whose input is selected as continuous or gated. In the continuous mode, the OPI is on and VS passes the signal to A1. In the gated mode, the OPI is turned on and off by the presence or absence of a --12 V gate pulse. The optical coupler is used as a convenient level shifter and provides a means of isolating the iontophoresis system ground from the ground of the digital timing device. One-third of the triple SPDT analog voltage switch is used for each of the 3 pumping channels. The control amplifier for the balance channel (not shown in Fig. 2) has two DC-only inputs, plus an input from each of the other three control amplifier outputs. The input resistors for these 3 inputs are trimmed so that the fourth current source produces an equal but opposite polarity current to the sum of the currents of the 3 pumping channels.
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Fig. 2. (A) control amplifier, (B) current source and high-voltage regulators, (C) current monitor and (D) unbalance current monitor. A1, A4, A5, Precision Monolithics OP-05CJ with offset trim; VS, RCS CD4053AE; OPI, Hewlett Packard 5082-4350; A2, A3, Analog Devices 171J; VR1, Motorola MC7812CP; VR2, Motorola MC7912CP, A6, A7, Fairchild pA741C; C1, C2, National LM311N; M, Triplett 120-1060343. All resistors are -+1% tolerance unless marked. A1, A4, A5, A6 and A7 are operated with -+15 V power supplies. The current source in Fig. 2 (B) is derived from the configuration developed , by Colburn and Schwartz (1972), implemented here with high voltage amplifiers A2 and A3. Dreyer and Peper (1973) used
a capacitance-neutralized version of this circuit for single-channel, short-duration iontophoretic application of acetylcholine with a virtual ground current-tovoltage converter as the current monitor.
IONTOPHORESIS SYSTEM T h e c u r r e n t , I0, delivered t o t h e m i c r o p i p e t t e b y this circuit is
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and, for t h e values s h o w n , a scale f a c t o r of 2 0 0 n A / V is o b t a i n e d . A small value o f R x is desirable t o m a i n tain the high voltage c o m p l i a n c e available a t Vx, b u t this r e d u c e s t h e effective o u t p u t i m p e d a n c e set b y t h e c o m m o n - m o d e i n p u t i m p e d a n c e o f A3. With a c o m p r o m i s e value of 50 M ~ a n d each R1 a n d R 2 pair m a t c h e d t o 0.01%, t h e effective low f r e q u e n c y outp u t i m p e d n a c e o f t h e c u r r e n t source is a p p r o x i m a t e l y 5 × 101° ~ . T h e c u r r e n t m o n i t o r described b e l o w d e p e n d s o n t h e o u t p u t o f voltage f o l l o w e r A3 b e i n g an a c c u r a t e r e p r e s e n t a t i o n o f t h e voltage at t h e j u n c t i o n o f R x a n d t h e m i c r o p i p e t t e . This is t h e ease if t h e linear r a n g e of i n p u t voltage o f A3 is n o t e x c e e d e d . U n f o r t u n a t e l y , a c o m m o n c h a r a c t e r i s t i c of t h e available high voltage o p e r a t i o n a l amplifiers (e.g., T e l e d y n e P h i l b r i e k 1 0 2 2 a n d B u r r - B r o w n 3 5 8 2 J M , as well as t h e A n a l o g Devices 1 7 1 J used h e r e ) is t h a t t h e i r o u t p u t s a t u r a t i o n voltage is t y p i c a l l y 5 V greater t h a n t h e i r linear range of i n p u t voltage. T h u s , if A2 a n d A3 have c o m m o n p o w e r supplies a n d A2 saturates, t h e n t h e i n p u t voltage o f A3 c a n e x c e e d its linear range. This p r o b l e m is avoided a n d a 5 V safety m a r g i n is o b t a i n e d b y decreasing t h e m a g n i t u d e o f t h e dual p o w e r s u p p l y for A 2 t o 10 V b e l o w t h a t for A3. T w o dual supplies, each w i t h c u r r e n t c a p a c i t y for 4 amplifiers c a n be used, or a single dual s u p p l y w i t h t w i c e t h a t c a p a c i t y c a n be used w i t h t h e low-voltage IC r e g u l a t o r s ( V R 1 , V R 2 ) t o g e n e r a t e t h e s e c o n d set o f supply voltages. As s h o w n , V R 1 a n d V R 2 are plus a n d m i n u s 12 V regulators, respectively, a n d each R3 resistor is a series c h a i n of seven 0.5 W resistors, six of w h i c h are 1 K ~ . T h e s e v e n t h is selected t o give t h e desired 115 V o u t p u t . T h e c u r r e n t delivered t o t h e m i e r o p i p e t t e c a n be m o n i t o r e d as t h e voltage across the k n o w n resistor Rx. With t h e i n t e g r i t y of V0 g u a r a n t e e d even w h e n A2 s a t u r a t e s , t h e voltage across R x is V x - - V 0. T h e c u r r e n t m o n i t o r circuit in Fig. 2 (C) divides t h e Vx--V 0 d i f f e r e n c e by 20 so t h a t t h e c o m m o n - m o d e voltage limit o f A4 c a n n o t be exceeded. By m a t c h i n g t h e like values in each R4 a n d R5 pair t o 0.01%, t h e c o m m o n - m o d e r e j e c t i o n of t h e differential a m p l i f i e r circuit is s u f f i c i e n t to resolve 1 n A (Vx--V 0 = 50 m V ) w i t h a c o m m o n - m o d e voltage of 1 1 0 V. T h e o u t p u t of A4 can drive a strip c h a r t r e c o r d e r at 2.5 m V / n A a n d a _+500 p A m e t e r m o v e m e n t sealed to r e a d _+500 n A full scale. T h e a m o u n t o f u n b l a n c e c u r r e n t is m o n i t o r e d b y s u m m i n g t h e o u t p u t s o f t h e 4 individual c u r r e n t m o n itors w i t h a m p l i f i e r A5. C o m p a r a t o r s C1 a n d C2 c o m pare t h e a m p l i t u d e a n d sign o f this value w i t h t h e
401 a p p r o p r i a t e p o l a r i t y of a selectable r e f e r e n c e voltage. If the u n b a l a n c e c u r r e n t is greater t h a n t h e s e l e c t e d limit, t h e n o n e of t h e c o m p a r a t o r s will t u r n o n a led, i n d i c a t i n g a n e t c u r r e n t flow t o g r o u n d (+unbalance) or a n e t flow f r o m g r o u n d (--unbalance). T o achieve 1% or b e t t e r a c c u r a c y in t h e u n b a l a n c e c u r r e n t value, t h e i n p u t resistors to A5 usually n e e d t o be e i t h e r selected or t r i m m e d t o c o m p e n s a t e for t h e a c c u m u l a t i o n of small gain errors in t h e c o n t r o l amplifiers, t h e c u r r e n t sources, a n d t h e c u r r e n t m o n i t o r s .
Summary A circuit d e s c r i p t i o n for a m u l t i c h a n n e l c o n s t a n t c u r r e n t s o u r c e for m i c r o i o n t o p h o r e s i s is p r e s e n t e d , T h e c i r c u i t r y allows a high voltage c o m p l i a n c e , accurate m o n i t o r i n g o f i o n o t o p h o r e t i c c u r r e n t w i t h o u t use of batteries, a n d a u t o m a t i c c u r r e n t n e u t r a l i z a t i o n . Drugs m a y be applied e i t h e r m a n u a l l y or w i t h a n e x t e r n a l digital gate signal. A m o n i t o r to d e t e c t curr e n t u n b a l a n c e greater t h a n a p r e s e l e c t e d level is also included.
R~sum4
Systeme synchronise, de haut voltage pour iontophorese, avec contr61e precis du courant Les a u t e u r s d~crivent u n circuit de source de cour a n t c o n s t a n t ~ plusieurs c a n a u x p o u r la m i c r o i o n t o phor~se. Ce m o n t a g e p e r m e t u n e g r a n d e stabilit~ de voltage, u n contr61e precis du c o u r a n t i o n t o p h o r ~ t i q u e sans r e c o u r s ~ des piles et u n e n e u t r a l i s a t i o n a u t o m a t i q u e du c o u r a n t . Les drogues p e u v e n t ~tre injectdes m a n u e l l e m e n t ou ~ l'aide d ' u n signal b i n a i r e de d ~ c l e n c h e m e n t . U n systSme de contr61e qui perm e t de d ~ t e c t o r t o u t d~s~quilibre de c o u r a n t qui d~passe u n niveau choisi ~ l ' a v a n c e est ~ g a l e m e n t inclus.
References
Bradshaw, C.M., Szabadi, E. a n d R o b e r t s , M.H.T. T h e reflection of injection and retaining currents in the t i m e c o u r s e o f n e u r o n a l r e s p o n s e s to m i c r o e l e c t r o p h o r e t i c a l l y applied drugs. J. P h a r m . P h a r m a c o l . , 1973, 25: 5 1 3 - - 5 2 0 . C o l b u r n , T.R., a n d S c h w a r t z , E.A. L i n e a r voltage c o n t r o l of c u r r e n t passed t h r o u g h a m i c r o p i p e t t e w i t h variable resistance. Med. Biol. Eng., 1 9 7 2 , 10: 504--509. Dreyer, F. a n d Peper, K. I o n t o p h o r e t i c a p p l i c a t i o n o f a c e t y l c h o l i n e : a d v a n t a g e s of high resistance microp i p e t t e s in c o n j u n c t i o n w i t h an e l e c t r o n i c c u r r e n t
402 pump. Pflugers Arch. ges. Physiol., 1974, 348: 263--272. Freedman, R., Hoffer, B.J. and Woodward, D.J. A quantitative microiontophoretic analysis of responses of central neurons to noradrenaline: interactions with cobalt, manganese, verapamil and dichloroisoprenaline, Brit. J. Pharmacol., 1975, 54: 529--539. Geller, H.M. and Woodward, D.J. An improved constant current source for micro-iontophoretic drug application studies. Electroenceph. clin. Neurophysiol., 1972, 33: 430--432. Hockman, C.H. and Bieger, D. Chemical transmission in the mammalian central nervous system. Univer-
B.M. SMITH, B.J. HOFFER sity Park Press, Baltimore, 1976: 442. Hoffer, B.J., Neff, N.H. and Siggins, G.R. Release of norepinephrine from micropipettes. J. Neuropharmacol.. 1971a, 10: 175--180. Hoffer, B.J., Siggins, G.R. and Bloom, F.E. Studies on norepinephrine-containingafferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis. Brain Res., 1971b, 25: 523--534. Teschemacher, H., Opheim, K., Cox, B. and Goldstein, A. (1975). A peptide-like substance from pituitary that acts like morphone. I. Isolation, Life Sci., 1975, 16: 1771--1775.
398
© E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers, Ltd. Technical
contribution
A GATED, HIGH MONITORING
VOLTAGE
IONTOPHORESIS
SYSTEM
WITH
ACCURATE
CURRENT
B.M. S M I T H a n d B.J. H O F F E R *
Section on Technical Development, National Institu te o f Mental Health and National Institute o f Neurological and Communicative Disorders and Stroke, Bethesda, Md. 20014 and Laboratory o f Neuropharmacology, National Institute o f Mental Health, St. Elizabeth's Hospital, Washington, D.C. 20032 (U.S.A.) ( A c c e p t e d for p u b l i c a t i o n : J u n e 15, 1 9 7 7 )
T h e local a p p l i c a t i o n o f drugs f r o m m u l t i b a r r e l m i c r o p i p e t t e s b y m i c r o i o n t o p h o r e s i s has p r o v e n t o be o f great i m p o r t a n c e in e l u c i d a t i n g drug a c t i o n s o n specific areas of t h e n e r v o u s s y s t e m . By c i r c u m v e n t ing m a n y o f t h e m e c h a n i c a l a n d e n z y m a t i c barriers p r e s e n t in t h e b r a i n a n d m i n i m i z i n g r e m o t e or indir e c t d r u g a c t i o n , this t e c h n i q u e has allowed precise c h a r a c t e r i z a t i o n of a n u m b e r o f p u t a t i v e n e u r o t r a n s m i t t e r s ( H o c k m a n a n d Bieger 1 9 7 6 ) , as well as pharm a c o l o g i c a l agents w h i c h i n t e r a c t w i t h discrete classes o f synapses. As w i t h m a n y i n n o v a t i o n s , it h a s b e c o m e a p p a r e n t t h a t m i c r o i o n t o p h o r e s i s possesses its o w n p a r t i c u l a r set of t e c h n i c a l l i m i t a t i o n s a n d pot e n t i a l artifacts. A m o n g t h e m o s t i m p o r t a n t are: (1) failure of t h e p i p e t t e to release drug ( H o f f e r e t al. 1 9 7 1 a ) , (2) p o l a r i z a t i o n of the n e u r o n f r o m i o n t o p h o r e t i c c u r r e n t ( H o f f e r et al. 1 9 7 1 b ) a n d ( 3 ) a l t e r a t i o n s in a m o u n t of drug released due to c h a n g e s in t h e t i m e b e t w e e n d r u g e j e c t i o n s ( B r a d s h a w e t al. 1 9 7 3 ; F r e e d m a n e t al. 1 9 7 5 ) . This p a p e r describes an i o n t o p h o r e s i s s y s t e m t h a t p r o d u c e s a c c u r a t e drug e j e c t i o n a n d allows p r o p e r i n t e r p r e t a t i o n o f drug r e s p o n s e s b y p r o v i d i n g m o n i t o r s o f critical i o n t o p h o r e t i c p a r a m e t e r s . T h e s e novel f e a t u r e s are incorp o r a t e d i n t o an i n s t r u m e n t t h a t has t h e same general c o n f i g u r a t i o n as t h a t d e s c r i b e d b y Geller a n d Woodw a r d ( 1 9 7 2 ) : t h r e e c u r r e n t s o u r c e s p e r m i t applicat i o n o f t h r e e s e p a r a t e drugs while a f o u r t h b a l a n c e c h a n n e l m i n i m i z e s n e t c u r r e n t flow to g r o u n d . A c c u r a t e drug a p p l i c a t i o n t h r o u g h the m u l t i b a r r e l m i c r o p i p e t t e , w h o s e resistances can be several h u n d r e d m e g o h m s a n d o f t e n s h o w m a r k e d variability w i t h time a n d c u r r e n t passage, is p r o d u c e d by solids t a t e v o l t a g e - c o n t r o l l e d c u r r e n t sources w i t h high outp u t i m p e d a n c e a n d high voltage c o m p l i a n c e . In addition, t h e g e n e r a t i o n of the drug c u r r e n t for each o f
* P r e s e n t address: Dept. of P h a r m a c o l o g y , University of C o l o r a d o S c h o o l of Medicine, Denver, Colo. 8 0 2 2 0 , U.S.A.
t h e t h r e e p u m p i n g c h a n n e l s m a y be s y n c h r o n o u s l y c o n t r o l l e d b y a digital gate signal f r o m an e x t e r n a l t i m i n g device. This facilitates b o t h regular, periodic drug e j e c t i o n a n d c o m p u t e r analysis o f n e u r o n a l r e s p o n s e s ( F r e e d m a n e t al. 1975). T h e critical p a r a m e t e r of i n t e r e s t in i o n t o p h o r e s i s is t h e drug c u r r e n t actually b e i n g delivered t h r o u g h t h e m i c r o p i p e t t e . With a v o l t a g e - c o n t r o l l e d c u r r e n t s o u r c e p r o v i d i n g t h e c u r r e n t , the c o n t r o l voltage is generally used as t h e c u r r e n t m o n i t o r . However, with t h e n e e d to pass relatively large c u r r e n t s t h r o u g h high resistance p i p e t t e s , t h e p r o d u c t of c u r r e n t a n d resist a n c e c a n e x c e e d the voltage c o m p l i a n c e of the curr e n t source. W h e n this h a p p e n s , t h e linearity b e t w e e n c u r r e n t a n d c o n t r o l voltage ceases a n d the c o n t r o l voltage gives a false i n d i c a t i o n of drug c u r r e n t . T h e c u r r e n t s o u r c e design p r e s e n t e d here p r o v i d e s a m e a n s o f g e n e r a t i n g a c u r r e n t m o n i t o r t h a t gives a true reading even w h e n the voltage c o m p l i a n c e o f t h e c u r r e n t s o u r c e is e x c e e d e d . This t y p e of c u r r e n t m o n i t o r i n g r e d u c e s false negative findings of drug efficacy a n d p e r m i t s the inclusion of an u n b a l a n c e c u r r e n t m o n i t o r . T h e u n b a l a n c e m o n i t o r i n d i c a t e s w h e n the difference b e t w e e n the t o t a l drug c u r r e n t f r o m t h e t h r e e p u m p i n g barrels a n d t h e c u r r e n t f r o m the b a l a n c e barrel e x c e e d s a preset value. A n y s i g n i f i c a n t u n b a l a n c e generally indicates t h a t the voltage c o m p l i a n c e of o n e of t h e c u r r e n t s o u r c e s has b e e n r e a c h e d following a partial blockage of t h e c o r r e s p o n d i n g barrel of t h e m i c r o p i p e t t e . Moreover, this u n b a l a n c e alerts the e x p e r i m e n t e r t h a t a n y o b s e r v e d n e u r o n a l r e s p o n s e s m a y be due to electronic effects of the iontophoretic current, rather t h a n to the drug. A typical e x p e r i m e n t a l a p p l i c a t i o n is s h o w n in Fig. 1. I o n t o p h o r e s i s of 55 n A o f 13-endorphine ( E 5 5 ) , a 31 a m i n o acid p o l y p e p t i d e believed to be an endogen o u s o p i a t e ( T e s c h e m a c h e r e t al. 1 9 7 5 ) , p r o d u c e d a r e p r o d u c i b l e i n h i b i t i o n of firing of this rat c a u d a t e n e u r o n . T h e effects of 13-endorphine are a n t a g o n i z e d by i o n t o p h o r e s i s of 10 n A of the o p i a t e a n t a g o n i s t ,
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3o ";";i Fig. 1. Effects of iontophoresis ~-endorphine and naloxone (N) on spontaneous activity of a single caudate neuron in rat. The ratemeter record is continuous in time. The magnitude of the ejection current in nanoamperes is indicated by the numbers after each drug and the duration of ejection, by the underlying bars. Note that ~-endorphine elicits a powerful depression of spontaneous discharge rate, and this response is reversibly antagonized by naloxone. Similar interactions were seen in all six caudate neurons tested. Initial drug barrel resistances for these substances ranged from 75 to 300 M ~ .
naloxone (N10). This experiment illustrates a number of the advantages of the circuitry described here. First, utilization of the gate input to eject ~-endorphine at regular intervals produces uniform control inhibitions. Second, the high compliance of the pump plays an important role, no doubt, in obtaining prompt neuronal responses to this large 31 amino acid polypeptide with high drug barrel resistance. Finally, unbalance, in this experiment, was less than 10 nA, minimizing any possibility of electrotonic artifacts.
Circuit description The principal circuit components of the system are divided into 4 sections in Fig. 2: (A) control voltage amplifier; (B) current source and high-voltage regulators; (C) current monitor, and (D) unbalance current monitor. Amplifier A1 combines 3 input voltages to generate the negative control voltage --V c for the current source. The 3 inputs are (1) a DC voltage to generate a drug-holding current, (2) a second DC voltage for drug-pumping current which may be applied continuously by manual switch or pulsed on by an external
digital gate input and (3) an analog input used in place of (2) when a pulse generator with multiple calibrated outputs is available. The value of the DC 'holding' input is set by the 1K, 10-turn potentiometer, the 5 or 50 nA/turn range switch, and the polarity switch. The second DC voltage is generated in exactly the same manner but must pass through the solidstate analog switch VS. VS is controlled by the optical isolator OPI whose input is selected as continuous or gated. In the continuous mode, the OPI is on and VS passes the signal to A1. In the gated mode, the OPI is turned on and off by the presence or absence of a --12 V gate pulse. The optical coupler is used as a convenient level shifter and provides a means of isolating the iontophoresis system ground from the ground of the digital timing device. One-third of the triple SPDT analog voltage switch is used for each of the 3 pumping channels. The control amplifier for the balance channel (not shown in Fig. 2) has two DC-only inputs, plus an input from each of the other three control amplifier outputs. The input resistors for these 3 inputs are trimmed so that the fourth current source produces an equal but opposite polarity current to the sum of the currents of the 3 pumping channels.
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Fig. 2. (A) control amplifier, (B) current source and high-voltage regulators, (C) current monitor and (D) unbalance current monitor. A1, A4, A5, Precision Monolithics OP-05CJ with offset trim; VS, RCS CD4053AE; OPI, Hewlett Packard 5082-4350; A2, A3, Analog Devices 171J; VR1, Motorola MC7812CP; VR2, Motorola MC7912CP, A6, A7, Fairchild pA741C; C1, C2, National LM311N; M, Triplett 120-1060343. All resistors are -+1% tolerance unless marked. A1, A4, A5, A6 and A7 are operated with -+15 V power supplies. The current source in Fig. 2 (B) is derived from the configuration developed , by Colburn and Schwartz (1972), implemented here with high voltage amplifiers A2 and A3. Dreyer and Peper (1973) used
a capacitance-neutralized version of this circuit for single-channel, short-duration iontophoretic application of acetylcholine with a virtual ground current-tovoltage converter as the current monitor.
IONTOPHORESIS SYSTEM T h e c u r r e n t , I0, delivered t o t h e m i c r o p i p e t t e b y this circuit is
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and, for t h e values s h o w n , a scale f a c t o r of 2 0 0 n A / V is o b t a i n e d . A small value o f R x is desirable t o m a i n tain the high voltage c o m p l i a n c e available a t Vx, b u t this r e d u c e s t h e effective o u t p u t i m p e d a n c e set b y t h e c o m m o n - m o d e i n p u t i m p e d a n c e o f A3. With a c o m p r o m i s e value of 50 M ~ a n d each R1 a n d R 2 pair m a t c h e d t o 0.01%, t h e effective low f r e q u e n c y outp u t i m p e d n a c e o f t h e c u r r e n t source is a p p r o x i m a t e l y 5 × 101° ~ . T h e c u r r e n t m o n i t o r described b e l o w d e p e n d s o n t h e o u t p u t o f voltage f o l l o w e r A3 b e i n g an a c c u r a t e r e p r e s e n t a t i o n o f t h e voltage at t h e j u n c t i o n o f R x a n d t h e m i c r o p i p e t t e . This is t h e ease if t h e linear r a n g e of i n p u t voltage o f A3 is n o t e x c e e d e d . U n f o r t u n a t e l y , a c o m m o n c h a r a c t e r i s t i c of t h e available high voltage o p e r a t i o n a l amplifiers (e.g., T e l e d y n e P h i l b r i e k 1 0 2 2 a n d B u r r - B r o w n 3 5 8 2 J M , as well as t h e A n a l o g Devices 1 7 1 J used h e r e ) is t h a t t h e i r o u t p u t s a t u r a t i o n voltage is t y p i c a l l y 5 V greater t h a n t h e i r linear range of i n p u t voltage. T h u s , if A2 a n d A3 have c o m m o n p o w e r supplies a n d A2 saturates, t h e n t h e i n p u t voltage o f A3 c a n e x c e e d its linear range. This p r o b l e m is avoided a n d a 5 V safety m a r g i n is o b t a i n e d b y decreasing t h e m a g n i t u d e o f t h e dual p o w e r s u p p l y for A 2 t o 10 V b e l o w t h a t for A3. T w o dual supplies, each w i t h c u r r e n t c a p a c i t y for 4 amplifiers c a n be used, or a single dual s u p p l y w i t h t w i c e t h a t c a p a c i t y c a n be used w i t h t h e low-voltage IC r e g u l a t o r s ( V R 1 , V R 2 ) t o g e n e r a t e t h e s e c o n d set o f supply voltages. As s h o w n , V R 1 a n d V R 2 are plus a n d m i n u s 12 V regulators, respectively, a n d each R3 resistor is a series c h a i n of seven 0.5 W resistors, six of w h i c h are 1 K ~ . T h e s e v e n t h is selected t o give t h e desired 115 V o u t p u t . T h e c u r r e n t delivered t o t h e m i e r o p i p e t t e c a n be m o n i t o r e d as t h e voltage across the k n o w n resistor Rx. With t h e i n t e g r i t y of V0 g u a r a n t e e d even w h e n A2 s a t u r a t e s , t h e voltage across R x is V x - - V 0. T h e c u r r e n t m o n i t o r circuit in Fig. 2 (C) divides t h e Vx--V 0 d i f f e r e n c e by 20 so t h a t t h e c o m m o n - m o d e voltage limit o f A4 c a n n o t be exceeded. By m a t c h i n g t h e like values in each R4 a n d R5 pair t o 0.01%, t h e c o m m o n - m o d e r e j e c t i o n of t h e differential a m p l i f i e r circuit is s u f f i c i e n t to resolve 1 n A (Vx--V 0 = 50 m V ) w i t h a c o m m o n - m o d e voltage of 1 1 0 V. T h e o u t p u t of A4 can drive a strip c h a r t r e c o r d e r at 2.5 m V / n A a n d a _+500 p A m e t e r m o v e m e n t sealed to r e a d _+500 n A full scale. T h e a m o u n t o f u n b l a n c e c u r r e n t is m o n i t o r e d b y s u m m i n g t h e o u t p u t s o f t h e 4 individual c u r r e n t m o n itors w i t h a m p l i f i e r A5. C o m p a r a t o r s C1 a n d C2 c o m pare t h e a m p l i t u d e a n d sign o f this value w i t h t h e
401 a p p r o p r i a t e p o l a r i t y of a selectable r e f e r e n c e voltage. If the u n b a l a n c e c u r r e n t is greater t h a n t h e s e l e c t e d limit, t h e n o n e of t h e c o m p a r a t o r s will t u r n o n a led, i n d i c a t i n g a n e t c u r r e n t flow t o g r o u n d (+unbalance) or a n e t flow f r o m g r o u n d (--unbalance). T o achieve 1% or b e t t e r a c c u r a c y in t h e u n b a l a n c e c u r r e n t value, t h e i n p u t resistors to A5 usually n e e d t o be e i t h e r selected or t r i m m e d t o c o m p e n s a t e for t h e a c c u m u l a t i o n of small gain errors in t h e c o n t r o l amplifiers, t h e c u r r e n t sources, a n d t h e c u r r e n t m o n i t o r s .
Summary A circuit d e s c r i p t i o n for a m u l t i c h a n n e l c o n s t a n t c u r r e n t s o u r c e for m i c r o i o n t o p h o r e s i s is p r e s e n t e d , T h e c i r c u i t r y allows a high voltage c o m p l i a n c e , accurate m o n i t o r i n g o f i o n o t o p h o r e t i c c u r r e n t w i t h o u t use of batteries, a n d a u t o m a t i c c u r r e n t n e u t r a l i z a t i o n . Drugs m a y be applied e i t h e r m a n u a l l y or w i t h a n e x t e r n a l digital gate signal. A m o n i t o r to d e t e c t curr e n t u n b a l a n c e greater t h a n a p r e s e l e c t e d level is also included.
R~sum4
Systeme synchronise, de haut voltage pour iontophorese, avec contr61e precis du courant Les a u t e u r s d~crivent u n circuit de source de cour a n t c o n s t a n t ~ plusieurs c a n a u x p o u r la m i c r o i o n t o phor~se. Ce m o n t a g e p e r m e t u n e g r a n d e stabilit~ de voltage, u n contr61e precis du c o u r a n t i o n t o p h o r ~ t i q u e sans r e c o u r s ~ des piles et u n e n e u t r a l i s a t i o n a u t o m a t i q u e du c o u r a n t . Les drogues p e u v e n t ~tre injectdes m a n u e l l e m e n t ou ~ l'aide d ' u n signal b i n a i r e de d ~ c l e n c h e m e n t . U n systSme de contr61e qui perm e t de d ~ t e c t o r t o u t d~s~quilibre de c o u r a n t qui d~passe u n niveau choisi ~ l ' a v a n c e est ~ g a l e m e n t inclus.
References
Bradshaw, C.M., Szabadi, E. a n d R o b e r t s , M.H.T. T h e reflection of injection and retaining currents in the t i m e c o u r s e o f n e u r o n a l r e s p o n s e s to m i c r o e l e c t r o p h o r e t i c a l l y applied drugs. J. P h a r m . P h a r m a c o l . , 1973, 25: 5 1 3 - - 5 2 0 . C o l b u r n , T.R., a n d S c h w a r t z , E.A. L i n e a r voltage c o n t r o l of c u r r e n t passed t h r o u g h a m i c r o p i p e t t e w i t h variable resistance. Med. Biol. Eng., 1 9 7 2 , 10: 504--509. Dreyer, F. a n d Peper, K. I o n t o p h o r e t i c a p p l i c a t i o n o f a c e t y l c h o l i n e : a d v a n t a g e s of high resistance microp i p e t t e s in c o n j u n c t i o n w i t h an e l e c t r o n i c c u r r e n t
402 pump. Pflugers Arch. ges. Physiol., 1974, 348: 263--272. Freedman, R., Hoffer, B.J. and Woodward, D.J. A quantitative microiontophoretic analysis of responses of central neurons to noradrenaline: interactions with cobalt, manganese, verapamil and dichloroisoprenaline, Brit. J. Pharmacol., 1975, 54: 529--539. Geller, H.M. and Woodward, D.J. An improved constant current source for micro-iontophoretic drug application studies. Electroenceph. clin. Neurophysiol., 1972, 33: 430--432. Hockman, C.H. and Bieger, D. Chemical transmission in the mammalian central nervous system. Univer-
B.M. SMITH, B.J. HOFFER sity Park Press, Baltimore, 1976: 442. Hoffer, B.J., Neff, N.H. and Siggins, G.R. Release of norepinephrine from micropipettes. J. Neuropharmacol.. 1971a, 10: 175--180. Hoffer, B.J., Siggins, G.R. and Bloom, F.E. Studies on norepinephrine-containingafferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis. Brain Res., 1971b, 25: 523--534. Teschemacher, H., Opheim, K., Cox, B. and Goldstein, A. (1975). A peptide-like substance from pituitary that acts like morphone. I. Isolation, Life Sci., 1975, 16: 1771--1775.
