- Email: [email protected]
Constant current source for iontophoresis
JournalofNeuroscience Methods, 29 (1989) 85-89 Elsevier
85
NSM00966
Constant current source for iontophoresis M.R. Park Department of Anatomy and Neurobiology, University of Tennessee. Memphis, TN 38163 (U.S.A.) (Received 5 July 1988) (Revised 23 January 1989) (Accepted 4 February 1989)
Key words: I o n t o p h o r e s i s ; C u r r e n t i n j e c t i o n ; C o n s t a n t c u r r e n t a m p l i f i e r ; I n s t r u m e n t a t i o n : T r a c t - t r a c i n g m e t h o d ; Phaseolus vulgaris l e u c o a g g l u t i n i n A simple, battery-powered current source is described that is suitable for the iontophoresis of axonal tracing substances, such as Phaseolus culgaris leucoagglutinin. Unlike the previous designs that form the basis of most commonly used iontophoretic devices, this circuit does not use operational amplifiers to provide controlled current. Instead, a pair of bipolar transistors that can be selected to provide a compliance of many hundreds of volts form the regulating circuitry.
Introduction Amplifiers that are used to deliver constant c u r r e n t s are e m p l o y e d extensively in neuroscience. I n c u r r e n t r a n g e s of 0 - 1 0 n A , they are u s e d for the i n t r a c e l l u l a r i n j e c t i o n of tracers, such as h o r s e r a d i s h p e r o x i d a s e , a n d for the e x p e r i m e n t a l m a n i p u l a t i o n of m e m b r a n e p o t e n t i a l . C o n s t a n t c u r r e n t a m p l i f i e r s that o p e r a t e i n c u r r e n t r a n g e s 1 0 - 1 0 0 0 times larger are used for the e x t r a c e l l u l a r d e p o s i t i o n of tracers a n d p h a r m a c e u t i c a l agents. S o m e inventive circuits for c u r r e n t injection are used for these purposes. The H o w l a n d current pump (Fig. 1A) is a classic circuit in which controlled current can be obtained with a single operational amplifier. It has b e e n p o p u l a r i z e d in o u r field b y G e l l e r a n d W o o d w a r d (1972). E n g i n e e r s k n o w it as a w e l l - s t u d i e d exercise f r o m their stud e n t days b u t view it as a n i m p r a c t i c a l c i r c u i t ( H o r o w i t z a n d Hill, 1980, p. 97), h a v i n g the d r a w b a c k s that the r a t i o s of resistors that m a k e
Correspondence: M.R. Park, Department of Anatomy and Neurobiology, University of Tennessee, Memphis, 875 Monroe Ave., Memphis, TN 38163, U.S.A.
u p the n e g a t i v e a n d p o s i t i v e f e e d b a c k loops m u s t m a t c h e x a c t l y a n d , in a c t u a l use, it is p r o n e to oscillation. A s e c o n d t y p e of c o n s t a n t c u r r e n t
R5 A
¢2500 v~
R1 [ ~.. 1011-pf v i ~" " "~ ' ~ ' --' \0pf I t/
B
Vni~
Rs
~r -r--~- -nq~F ~ ~ I 22 M ;. 10 K /, 100 K ~ _1 ..]__ i _-.... -z-
Load
_i_
Fig. 1. Operational amplifier designs for controlled-current amplifiers. A: the Howland current pump requires just a single operational amplifier but a high-voltage type is required for iontophoretic applications. The ratio of resistors in the positive and negative feedback loops R1/R 5 = R z / ( R 3 + R4), must be precisely matched. B: the common bootstrap design used in active bridge amplifiers for intracellular recording can also be adopted as a current source by using high-voltage operational amplifiers. The design is simple and the finished circuit is stable and accurate. The schematized summing amplifier (upper of the two) would consist of at least two operational amplifiers in a practical circuit. A large fraction of the compliance otherwise offered by the high output voltage swings of the operational amplifiers is wasted as a voltage drop across R~.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
86 source, the active bridge amplifier (Fig. 1B), uses several (usually 3) operational amplifiers connected in a precisely adjusted feedback loop (Dreyer and Peper, 1974). This circuit was developed originally for intracellular current injection through recording micropipettes (Fein, 1966) and is the most c o m m o n circuit for intracellular recording amplifiers. As a pure current injection amplifier, as proposed by Dreyer and Peper (1974), it has the advantage of stability but the disadvantage of high cost due to the high voltage operational amplifiers that is uses. Moreover, the circuit wastes a major portion of its compliance due to the voltage drop across the current injection resistor, Rs. For m a n y applications, much less elaborate devices can be used. Yet, because the principles underlying the use of discrete components are not generally known to neuroscientists, the more complicated and expensive implementations prevail. It is possible to build high-compliance controlled-current sources that use just a few active components (diodes or transistors). Knowledge of how to do so should be part of the standard repertoire of techniques for neuroscientists, just as they are for electrical engineers.
Materials and Methods The impedance of the load limits the m a x i m u m current that can be injected by a constant current device. The quality that describes a current source's ability to inject current, even into high resistance loads, is called compliance. Compliance is expressed in volts and for iontophoretic devices it is usually in the range of hundreds of volts (e.g. from Ohm's Law, to pass 5/~A through a 100 MI2 pipette requires 500 V of compliance). Compliance can be no more than the supply voltage of the current-injecting device but a bad choice of circuitry can reduce it to well below this value. For example, the current delivered to the load in the active bridge amplifier shown in Fig. 1B also passes through the series resistor, Rs. R s typically has a value of 100 MJ2, which effectively wastes one-half of the potential compliance offered in the
A
Voc
B
.'---~to~d
a~
½
e
V~c
~Re
C
Vc~
,
[- i LR e
Fig. 2. Controlled-current sources using discrete components. If a constant voltage can be supplied to the base of a bipolar trat~,sistor, then a constant current roughly equal to that voltage divided by the value of the emitter resistor (I = l/b/Re) will be delivered across the load. A: the voltage divider formed by R~ and R 2 is one possible way of providing a constant Vb. In this case Vb= V ~ . R 2 / ( R 1 + R2). This circuit is not appropriate for battery-powered devices. B: a zener diode, Ze, is the classic voltage reference and provides a suitable way to deliver a controlled Vb. C: in this feedback design, Q2 m&intains Vb at a value such that the voltage across R e is 0.6 V. The current flowing through R e is essentially the current passing through the load and has the controlled value I = 0.6/R e.
previous example (i.e. 500 V power supply and a load of 100 M~2). The functioning of bipolar transistors in the constant current circuits depicted in Fig. 2 is treated in m a n y electronics references (e.g. Horowitz and Hill, 1980). Briefly, in each of the 3 circuits (Fig. 2), a positive voltage is applied to the base of the transistor, Q1. As long as the base of Q1 is positive with respect to its emitter, the transistor is said to be on. In this state, positive current flows from base to emitter and this enables a much larger current to flow from collector to emitter. The ratio of these two currents is the gain of the transistor, abbreviated h fe, and is typically between 30 and 300. These two currents sum (Ibe + I ~ ) and pass through the regulating resistor, Re, raising the voltage at the emitter by Ve = Re" (1be + I ~ ) . This in turn reduces the driving force for, 1be and it together with lee are reduced. The relationship is expressed as follows: Vb - 0.6 Re
/be + Ic~
Vb is the voltage at the transistor's base, 0.6 is the voltage drop between the base and emitter of silicon transistors, R e is the value of the emitter
87
15 k~ 51 k.Q
$1 pulse duration and frequency control +9v
1 M~
+180 V
6.8 k.Q
on/off $3
t
_2+ --~ 9V
22 k~
4 x 4 5 V -~"
39k.Q $2 pulse/constant (optional)
200 k~ 390 k~ +
-
m
10 I.Lf0v
$4 polarity I o
220 k~ R2 High Voltage Transistors Transistor* 2N5416 SK3747/157 ECG157 SK3045/225 SK3115/165
(
Vceo; Hfe 300 50 300 80 300 30 min 350 40 rain 1500 5 min
* PNP types only
o
I
current output
,~) 50p.A
Q1 (ECG 157) Q2 (ECG 157 Rc~
current amplitude control 500 k.Q log R1 10k.Q
0.6 I = Rc+ R1
Fig. 3. A practical battery-powered high compliance iontophoretic device. The feedback design in Fig. 2C is used to regulate current. The table lists a few suitable high-voltage transistors, their breakdown voltages (Vc~) and gain (Hfe). E C G transistors are Sylvania products and SK ones are from RCA. Transistors with 2N designations are made by m a n y manufacturers. The L555 integrated circuit is a simple timing device with very low current drain, making it suitable for a battery-powered device. It is wired to deliver square-wave pulses having a 50% duty cycle. The values of the timing resistors give periods of 0.2, 0.7, 1.2, 6, and 12 s. The power supply has two parts: a pack of 4 or more 45 V batteries (Eveready No. 415, N E D A No. 213) provides the high voltage and a single 9 V battery provides bias for the transistors and power for the L555.
8~
resistor, and /be and I c e a r e the base-emitter and collector-emitter currents, respectively. For typical values of h f~, /be can be neglected and we have the relation: Vb - 0.6 R,
1~
I ~ is the current passing through the load. Since both Vb and R~ can be regulated, we now have a way of delivering controlled current. The circuit in Fig. 2A controls Vb by means of a voltage divider, such that V b = Ve • R 2 / ( R 1 + R2). This is not a good choice for a batterypowered device as (1) it depends upon having a stable supply voltage, which is not the case as batteries discharge and (2) substantial current must flow through the voltage-dividing resistors, causing an unwanted drain on the battery supply. In Fig. 2B, a zener diode is used to provide a constant Vb. This will operate independently of supply voltage, so long as V~ is above the zener threshold, but it still presents an unneeded drain on the battery supply. The circuit shown in Fig. 2C differs in that the voltage across R~, rather than Vb, is directly regulated. Transistor Q2 serves to measure the voltage across R~. If it rises above 0.6 V, Q2 begins conducting a collector to emitter current, reducing the voltage at the base of Q1 and hence the current through R e. A negative feedback system has been formed in which Q2 is maintaining a constant 0.6 V across R e. Once again neglecting Ibe, the current flowing though the load is maintained at: 0.6 Re
Ice ~ --
Thus, the value of Re determines the magnitude of the current delivered by the device. The complete circuit is shown in Fig. 3. High voltage transistors are used for Q1 and Q2, allowing compliances of hundreds of volts (see Fig. 3 for a list of suitable transistors). A standard 555 integrated circuit timer, of a type with low-current demand (e.g. LM555 or I C M 7555 IPA), as is appropriate for battery power, is used tO deliver square-wave c o m m a n d pulses that turn the current regulating circuitry on and off. The arrangement and values of the timing resistors provide pulses
with a 50% duty cycle and periods of 12. 0, 1.0.7, and 0.2 s. The amplitude of the square wave pulses output by the 555 timer is termed ~] in the following discussion. With a fresh 9 V battery, V~ will be at or slightly above 9 V. As the battery discharges, I~ will sink to as low as 7 V without affecting the performance of the device. Timing is independent of supply voltage and the 9 V battery that supplies the timing circuitry, typically lasts for years. Current amplitude is controlled by the potentiometer, R~. The design should provided regulated current in the range of 1.2-60 /~A, based upon the extremes of emitter resistance (R c + R 1) permitted for the values shown. However, the values of R 1 and R c cannot be freely substituted in order to change the operating range of the device without also taking into account the role that R 2 plays in limiting both minimum and m a x i m u m currents. The value of R 2 limits the maximum current, I,na~, because it determines the maximum base current that can be delivered to Q1. I ....... = h f e i Q 1 ) V i / R 2
Assuming worst case values of 7 V and 30 for Vt and h fe(Ql), respectively, this becomes I .... = 30- 7 / R 2 = 2 0 0 / R 2 This fixes a m a x i m u m value of R z at about 200/Ima X which would be 3.3 M~2, a standard resistor value, for the/max of 60/~A already predicted. The current passed by R 2 cannot be too large, however, as Q2 is limited by its own gain as to how much current it can shunt from the base of Q1. The minimum value for R 2 is determined by the relationship
J/~/R2 <
hfe~Q2) " /rain
The appropriate worst case values are now 9 V and 30 for Vt and hf~Q2 ~, respectively, and give this rule: R 2 "( 0-2/Imin
which yields a minimum R z of 200 k~2 for the desired In~, of 1.2 p,A. The compliance of this device is essentially determined by the supply voltage. A pack of four
~9 45 V batteries, yielding a supply voltage of 180 V, has been adequate for our purposes. The n u m b e r of batteries used could be increased to provide quite large compliances, only taking care that the breakdown voltage, V~.... of the transistors chosen for Q1 and Q2 is not exceeded. Current is monitored by means of a meter. Using a battery power supply confers the advantage of not having a reference to any particular ground potential so that polarity reversal can be accomplished by simply switching the leads. The polarity switch, $4, accomplishes this. An optional switch, labeled $2 in the schematic, disconnects the timing circuitry so that a steady current is injected.
0.2 x 10 9 A, depending upon the length of time that the device has been on. This variation in the magnitude of the leak is a temperature effect and is expected in a device not having temperature compensation circuitry. This magnitude of leak current is insignificant in an instrument designed to operate in t h e / z A range. Devices built to this design have been principally used for the injection of Phaseolus vulgaris leucoagglutinin ( P H A - L ) in axonal tracing studies (e.g. Kita and Kitai, 1987: Behbehani et al., 1988). With a compliance of 180 V, currents in the order of 5 /~A can be successfully passed through the P H A - L pipettes that we use ( 1 5 - 3 0 btM tips, 2.5% P H A - L in 0.05 M sodium phosphate-buffered saline).
Discussion Acknowledgement The design goals were simplicity, reliability, and ease of use. W h a t has resulted is almost incidentally a low cost device. It is battery-powered so that most problems of grounding and electrical interference are eliminated. The feedback-constant current source utilizing a pair of bipolar transistors is more accurate than corresponding circuits based on field-effect transistors (FETs) (e.g. Horowitz and Hill, pp. 241-2). However, bipolar transistors have several properties that must be considered in the design and use of this device. One is that gain (h re) varies considerably a m o n g individual transistors of the same type. This design, like all correct bipolar transistor designs, does not depend u p o n a particular value of h f~. A second property is that bipolar transistors exhibit a small leak current that flows through collector and emitter when the transistor is in its off state. The leak current of the author's device, measured as the quiescent current flowing during the off part of the current injection cycle, was found to vary between 0.1 × 10 -9 A and
Supported by U S P H S G r a n t NS20841.
References Behbehani, M.M., Park, M.R. and Clement, M.E. (1988) lntcractions between the lateral hypothalamus and the periaqueductal gray, J. Neurosci., 8: 2780-2787. Dreyer, F. and Peper, K. (1974) lontophoretic application of acetylcholine: advantages of high resistance micropipettes in connection with an electronic current pump, Pflt~gers Arch., 348: 263-272. Fein, H. (1966) Passing current through recording glass micropipette electrodes, I EEE Trans. Biomed. Eng., 13: 211-212. Geller, H.M. and Woodward, D.J. (1972) An improved constant current source for microiontophoretic drug application studies, Electroencephalogr. Clin. Neurophysiol., 33: 430-432. Horowitz, P. and Hill, W. (1980) The Art of Electronics, Cambridge University, New York, pp. 97, 241-242. Kita. H. and Kitai, S.T. (1987) Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method, J. Comp. Neurol., 260:435 452.
85
NSM00966
Constant current source for iontophoresis M.R. Park Department of Anatomy and Neurobiology, University of Tennessee. Memphis, TN 38163 (U.S.A.) (Received 5 July 1988) (Revised 23 January 1989) (Accepted 4 February 1989)
Key words: I o n t o p h o r e s i s ; C u r r e n t i n j e c t i o n ; C o n s t a n t c u r r e n t a m p l i f i e r ; I n s t r u m e n t a t i o n : T r a c t - t r a c i n g m e t h o d ; Phaseolus vulgaris l e u c o a g g l u t i n i n A simple, battery-powered current source is described that is suitable for the iontophoresis of axonal tracing substances, such as Phaseolus culgaris leucoagglutinin. Unlike the previous designs that form the basis of most commonly used iontophoretic devices, this circuit does not use operational amplifiers to provide controlled current. Instead, a pair of bipolar transistors that can be selected to provide a compliance of many hundreds of volts form the regulating circuitry.
Introduction Amplifiers that are used to deliver constant c u r r e n t s are e m p l o y e d extensively in neuroscience. I n c u r r e n t r a n g e s of 0 - 1 0 n A , they are u s e d for the i n t r a c e l l u l a r i n j e c t i o n of tracers, such as h o r s e r a d i s h p e r o x i d a s e , a n d for the e x p e r i m e n t a l m a n i p u l a t i o n of m e m b r a n e p o t e n t i a l . C o n s t a n t c u r r e n t a m p l i f i e r s that o p e r a t e i n c u r r e n t r a n g e s 1 0 - 1 0 0 0 times larger are used for the e x t r a c e l l u l a r d e p o s i t i o n of tracers a n d p h a r m a c e u t i c a l agents. S o m e inventive circuits for c u r r e n t injection are used for these purposes. The H o w l a n d current pump (Fig. 1A) is a classic circuit in which controlled current can be obtained with a single operational amplifier. It has b e e n p o p u l a r i z e d in o u r field b y G e l l e r a n d W o o d w a r d (1972). E n g i n e e r s k n o w it as a w e l l - s t u d i e d exercise f r o m their stud e n t days b u t view it as a n i m p r a c t i c a l c i r c u i t ( H o r o w i t z a n d Hill, 1980, p. 97), h a v i n g the d r a w b a c k s that the r a t i o s of resistors that m a k e
Correspondence: M.R. Park, Department of Anatomy and Neurobiology, University of Tennessee, Memphis, 875 Monroe Ave., Memphis, TN 38163, U.S.A.
u p the n e g a t i v e a n d p o s i t i v e f e e d b a c k loops m u s t m a t c h e x a c t l y a n d , in a c t u a l use, it is p r o n e to oscillation. A s e c o n d t y p e of c o n s t a n t c u r r e n t
R5 A
¢2500 v~
R1 [ ~.. 1011-pf v i ~" " "~ ' ~ ' --' \0pf I t/
B
Vni~
Rs
~r -r--~- -nq~F ~ ~ I 22 M ;. 10 K /, 100 K ~ _1 ..]__ i _-.... -z-
Load
_i_
Fig. 1. Operational amplifier designs for controlled-current amplifiers. A: the Howland current pump requires just a single operational amplifier but a high-voltage type is required for iontophoretic applications. The ratio of resistors in the positive and negative feedback loops R1/R 5 = R z / ( R 3 + R4), must be precisely matched. B: the common bootstrap design used in active bridge amplifiers for intracellular recording can also be adopted as a current source by using high-voltage operational amplifiers. The design is simple and the finished circuit is stable and accurate. The schematized summing amplifier (upper of the two) would consist of at least two operational amplifiers in a practical circuit. A large fraction of the compliance otherwise offered by the high output voltage swings of the operational amplifiers is wasted as a voltage drop across R~.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
86 source, the active bridge amplifier (Fig. 1B), uses several (usually 3) operational amplifiers connected in a precisely adjusted feedback loop (Dreyer and Peper, 1974). This circuit was developed originally for intracellular current injection through recording micropipettes (Fein, 1966) and is the most c o m m o n circuit for intracellular recording amplifiers. As a pure current injection amplifier, as proposed by Dreyer and Peper (1974), it has the advantage of stability but the disadvantage of high cost due to the high voltage operational amplifiers that is uses. Moreover, the circuit wastes a major portion of its compliance due to the voltage drop across the current injection resistor, Rs. For m a n y applications, much less elaborate devices can be used. Yet, because the principles underlying the use of discrete components are not generally known to neuroscientists, the more complicated and expensive implementations prevail. It is possible to build high-compliance controlled-current sources that use just a few active components (diodes or transistors). Knowledge of how to do so should be part of the standard repertoire of techniques for neuroscientists, just as they are for electrical engineers.
Materials and Methods The impedance of the load limits the m a x i m u m current that can be injected by a constant current device. The quality that describes a current source's ability to inject current, even into high resistance loads, is called compliance. Compliance is expressed in volts and for iontophoretic devices it is usually in the range of hundreds of volts (e.g. from Ohm's Law, to pass 5/~A through a 100 MI2 pipette requires 500 V of compliance). Compliance can be no more than the supply voltage of the current-injecting device but a bad choice of circuitry can reduce it to well below this value. For example, the current delivered to the load in the active bridge amplifier shown in Fig. 1B also passes through the series resistor, Rs. R s typically has a value of 100 MJ2, which effectively wastes one-half of the potential compliance offered in the
A
Voc
B
.'---~to~d
a~
½
e
V~c
~Re
C
Vc~
,
[- i LR e
Fig. 2. Controlled-current sources using discrete components. If a constant voltage can be supplied to the base of a bipolar trat~,sistor, then a constant current roughly equal to that voltage divided by the value of the emitter resistor (I = l/b/Re) will be delivered across the load. A: the voltage divider formed by R~ and R 2 is one possible way of providing a constant Vb. In this case Vb= V ~ . R 2 / ( R 1 + R2). This circuit is not appropriate for battery-powered devices. B: a zener diode, Ze, is the classic voltage reference and provides a suitable way to deliver a controlled Vb. C: in this feedback design, Q2 m&intains Vb at a value such that the voltage across R e is 0.6 V. The current flowing through R e is essentially the current passing through the load and has the controlled value I = 0.6/R e.
previous example (i.e. 500 V power supply and a load of 100 M~2). The functioning of bipolar transistors in the constant current circuits depicted in Fig. 2 is treated in m a n y electronics references (e.g. Horowitz and Hill, 1980). Briefly, in each of the 3 circuits (Fig. 2), a positive voltage is applied to the base of the transistor, Q1. As long as the base of Q1 is positive with respect to its emitter, the transistor is said to be on. In this state, positive current flows from base to emitter and this enables a much larger current to flow from collector to emitter. The ratio of these two currents is the gain of the transistor, abbreviated h fe, and is typically between 30 and 300. These two currents sum (Ibe + I ~ ) and pass through the regulating resistor, Re, raising the voltage at the emitter by Ve = Re" (1be + I ~ ) . This in turn reduces the driving force for, 1be and it together with lee are reduced. The relationship is expressed as follows: Vb - 0.6 Re
/be + Ic~
Vb is the voltage at the transistor's base, 0.6 is the voltage drop between the base and emitter of silicon transistors, R e is the value of the emitter
87
15 k~ 51 k.Q
$1 pulse duration and frequency control +9v
1 M~
+180 V
6.8 k.Q
on/off $3
t
_2+ --~ 9V
22 k~
4 x 4 5 V -~"
39k.Q $2 pulse/constant (optional)
200 k~ 390 k~ +
-
m
10 I.Lf0v
$4 polarity I o
220 k~ R2 High Voltage Transistors Transistor* 2N5416 SK3747/157 ECG157 SK3045/225 SK3115/165
(
Vceo; Hfe 300 50 300 80 300 30 min 350 40 rain 1500 5 min
* PNP types only
o
I
current output
,~) 50p.A
Q1 (ECG 157) Q2 (ECG 157 Rc~
current amplitude control 500 k.Q log R1 10k.Q
0.6 I = Rc+ R1
Fig. 3. A practical battery-powered high compliance iontophoretic device. The feedback design in Fig. 2C is used to regulate current. The table lists a few suitable high-voltage transistors, their breakdown voltages (Vc~) and gain (Hfe). E C G transistors are Sylvania products and SK ones are from RCA. Transistors with 2N designations are made by m a n y manufacturers. The L555 integrated circuit is a simple timing device with very low current drain, making it suitable for a battery-powered device. It is wired to deliver square-wave pulses having a 50% duty cycle. The values of the timing resistors give periods of 0.2, 0.7, 1.2, 6, and 12 s. The power supply has two parts: a pack of 4 or more 45 V batteries (Eveready No. 415, N E D A No. 213) provides the high voltage and a single 9 V battery provides bias for the transistors and power for the L555.
8~
resistor, and /be and I c e a r e the base-emitter and collector-emitter currents, respectively. For typical values of h f~, /be can be neglected and we have the relation: Vb - 0.6 R,
1~
I ~ is the current passing through the load. Since both Vb and R~ can be regulated, we now have a way of delivering controlled current. The circuit in Fig. 2A controls Vb by means of a voltage divider, such that V b = Ve • R 2 / ( R 1 + R2). This is not a good choice for a batterypowered device as (1) it depends upon having a stable supply voltage, which is not the case as batteries discharge and (2) substantial current must flow through the voltage-dividing resistors, causing an unwanted drain on the battery supply. In Fig. 2B, a zener diode is used to provide a constant Vb. This will operate independently of supply voltage, so long as V~ is above the zener threshold, but it still presents an unneeded drain on the battery supply. The circuit shown in Fig. 2C differs in that the voltage across R~, rather than Vb, is directly regulated. Transistor Q2 serves to measure the voltage across R~. If it rises above 0.6 V, Q2 begins conducting a collector to emitter current, reducing the voltage at the base of Q1 and hence the current through R e. A negative feedback system has been formed in which Q2 is maintaining a constant 0.6 V across R e. Once again neglecting Ibe, the current flowing though the load is maintained at: 0.6 Re
Ice ~ --
Thus, the value of Re determines the magnitude of the current delivered by the device. The complete circuit is shown in Fig. 3. High voltage transistors are used for Q1 and Q2, allowing compliances of hundreds of volts (see Fig. 3 for a list of suitable transistors). A standard 555 integrated circuit timer, of a type with low-current demand (e.g. LM555 or I C M 7555 IPA), as is appropriate for battery power, is used tO deliver square-wave c o m m a n d pulses that turn the current regulating circuitry on and off. The arrangement and values of the timing resistors provide pulses
with a 50% duty cycle and periods of 12. 0, 1.0.7, and 0.2 s. The amplitude of the square wave pulses output by the 555 timer is termed ~] in the following discussion. With a fresh 9 V battery, V~ will be at or slightly above 9 V. As the battery discharges, I~ will sink to as low as 7 V without affecting the performance of the device. Timing is independent of supply voltage and the 9 V battery that supplies the timing circuitry, typically lasts for years. Current amplitude is controlled by the potentiometer, R~. The design should provided regulated current in the range of 1.2-60 /~A, based upon the extremes of emitter resistance (R c + R 1) permitted for the values shown. However, the values of R 1 and R c cannot be freely substituted in order to change the operating range of the device without also taking into account the role that R 2 plays in limiting both minimum and m a x i m u m currents. The value of R 2 limits the maximum current, I,na~, because it determines the maximum base current that can be delivered to Q1. I ....... = h f e i Q 1 ) V i / R 2
Assuming worst case values of 7 V and 30 for Vt and h fe(Ql), respectively, this becomes I .... = 30- 7 / R 2 = 2 0 0 / R 2 This fixes a m a x i m u m value of R z at about 200/Ima X which would be 3.3 M~2, a standard resistor value, for the/max of 60/~A already predicted. The current passed by R 2 cannot be too large, however, as Q2 is limited by its own gain as to how much current it can shunt from the base of Q1. The minimum value for R 2 is determined by the relationship
J/~/R2 <
hfe~Q2) " /rain
The appropriate worst case values are now 9 V and 30 for Vt and hf~Q2 ~, respectively, and give this rule: R 2 "( 0-2/Imin
which yields a minimum R z of 200 k~2 for the desired In~, of 1.2 p,A. The compliance of this device is essentially determined by the supply voltage. A pack of four
~9 45 V batteries, yielding a supply voltage of 180 V, has been adequate for our purposes. The n u m b e r of batteries used could be increased to provide quite large compliances, only taking care that the breakdown voltage, V~.... of the transistors chosen for Q1 and Q2 is not exceeded. Current is monitored by means of a meter. Using a battery power supply confers the advantage of not having a reference to any particular ground potential so that polarity reversal can be accomplished by simply switching the leads. The polarity switch, $4, accomplishes this. An optional switch, labeled $2 in the schematic, disconnects the timing circuitry so that a steady current is injected.
0.2 x 10 9 A, depending upon the length of time that the device has been on. This variation in the magnitude of the leak is a temperature effect and is expected in a device not having temperature compensation circuitry. This magnitude of leak current is insignificant in an instrument designed to operate in t h e / z A range. Devices built to this design have been principally used for the injection of Phaseolus vulgaris leucoagglutinin ( P H A - L ) in axonal tracing studies (e.g. Kita and Kitai, 1987: Behbehani et al., 1988). With a compliance of 180 V, currents in the order of 5 /~A can be successfully passed through the P H A - L pipettes that we use ( 1 5 - 3 0 btM tips, 2.5% P H A - L in 0.05 M sodium phosphate-buffered saline).
Discussion Acknowledgement The design goals were simplicity, reliability, and ease of use. W h a t has resulted is almost incidentally a low cost device. It is battery-powered so that most problems of grounding and electrical interference are eliminated. The feedback-constant current source utilizing a pair of bipolar transistors is more accurate than corresponding circuits based on field-effect transistors (FETs) (e.g. Horowitz and Hill, pp. 241-2). However, bipolar transistors have several properties that must be considered in the design and use of this device. One is that gain (h re) varies considerably a m o n g individual transistors of the same type. This design, like all correct bipolar transistor designs, does not depend u p o n a particular value of h f~. A second property is that bipolar transistors exhibit a small leak current that flows through collector and emitter when the transistor is in its off state. The leak current of the author's device, measured as the quiescent current flowing during the off part of the current injection cycle, was found to vary between 0.1 × 10 -9 A and
Supported by U S P H S G r a n t NS20841.
References Behbehani, M.M., Park, M.R. and Clement, M.E. (1988) lntcractions between the lateral hypothalamus and the periaqueductal gray, J. Neurosci., 8: 2780-2787. Dreyer, F. and Peper, K. (1974) lontophoretic application of acetylcholine: advantages of high resistance micropipettes in connection with an electronic current pump, Pflt~gers Arch., 348: 263-272. Fein, H. (1966) Passing current through recording glass micropipette electrodes, I EEE Trans. Biomed. Eng., 13: 211-212. Geller, H.M. and Woodward, D.J. (1972) An improved constant current source for microiontophoretic drug application studies, Electroencephalogr. Clin. Neurophysiol., 33: 430-432. Horowitz, P. and Hill, W. (1980) The Art of Electronics, Cambridge University, New York, pp. 97, 241-242. Kita. H. and Kitai, S.T. (1987) Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method, J. Comp. Neurol., 260:435 452.