Journal o f Neurosclence Methods, 1 (1979) 205--218 © Elsevier/North-Holland Biomedical Press

205

Research Papers SUSTAINED EXTRACELLULAR POTENTIALS CORD DURING THE MICROIONTOPHORETIC EXCITATORY AMINO ACIDS

IN THE CAT SPINAL APPLICATION OF

J.A. FLATMAN and J.D.C. LAMBERT Institute o f Physiology, Umverstty o f Aarhus, 8000 Aarhus C (Denmark) (Received April l l t h , 1979) (Accepted June 5th, 1979)

Sustained negative potentials were recorded in the ventral horn of the cat spinal cord during current balanced extracellular iontophoresls of excitatory amino acids. The potentials (referred to a distant indifferent electrode) were measured by an extracellular mlcroelectrode These focal potentials (FPs) were evoked by DL-homocysteate, L-glutamate, N-methyl-D-aspartate and kamate These FPs are not an artifact of extracellular mmroiontophoresis. Their time course is correlated with the depolarization of spinal motoneurones by excitatory amino acids. During iontophoresls of kainate, FPs can be as large as 50 mV and can be recorded for up to 1 mm from the site of drug application. The FP and depolarization caused by kainate were usually irreversible. The depolarization of motoneurones evoked by excitatory amino acids is very much larger when recorded as a 'transmembrane potential' (i.e. the potential of an intracellular electrode minus the potential of a local extracellular electrode) rather than as a 'classical' lntracellular potential (i e. referred to a distant reference electrode). Possible mechanisms for the generation of the FP are discussed. It is suggested that FP may be recorded routinely during microiontophoretic studies employing extracellular recording of neuronal activity. The apphcatlon of the FP as a measure of cell depolarization during pharmacological studies of excitatory amino acids and agents that block their action is discussed.

INTRODUCTION When drugs are applied to neurones by extracellular iontophoresis, interp r e t a t i o n o f t h e r e s u l t s o b t a i n e d is f r a u g h t w i t h d i f f i c u l t y ( B l o o m , 1 9 7 4 ) . M a n y o f t h e p r o b l e m s a r i s e f r o m t h e c h a n g e s in t h e e l e c t r i c a l a n d e l e c t r o chemical properties of the recording and iontophoretic microelectrodes during and after the passage of iontophoretic current. These problems have been extensively analyzed and reviewed by Krnjevi5 (1972). One particularly t r o u b l e s o m e a r t i f a c t is t h e r e c o r d i n g o f a s l o w l y i n c r e a s i n g D C p o t e n t i a l throughout the period of the microiontophoretic injection (KrnjeviS, 1972). We have recently made a detailed study of these slow DC potentials evoked b y m i c r o i o n t o p h o r e s i s o f a c t i v e a g e n t s in t h e a n t e r i o r h o r n o f t h e s p i n a l

206

cord of cats. These are not artifacts, but are related to the polarization of neurones in the vicinity of the recording electrode. METHODS

Thirteen cats (of both sexes 2.0--4.0 kg) were anaesthetized with either pentobarbitone (35 mg/kg intraperitoneally and then intravenously as required) or chloralose (50 mg/kg) and pentobarbitone (30 mg). Some cats were anaemically decerebrated following dissection under pentobarbitone anaesthesia (Engberg et al., 1979a). The animals were then paralyzed with gallamine and respired with intermittent positive pressure ventilation. Full details of the dissection and monitoring of blood pressure, respiration, and temperature, are given m Engberg et al. (1979a). Combined recording/iontophoretic units were used with a central single recording electrode surrounded by seven lontophoretic barrels (Engberg et al., 1979a). The recording and iontophoretic electrodes were mounted together on a single manipulator while the central recording electrode could be moved independently by a second mampulator (Fig. 1, and see Spehlmann, 1969). Both manipulators were driven by step drive motors (Eide and K~illstrSm, 1968). The combined electrode was assembled immediately before use. The recording electrode was moved through the iontophoretic assembly (under mmroscopic control) until its tip protruded by 12--36 pm. The electrode was then ready for tracking through the grey matter of the spinal cord. In later experiments we used fixed recording/iontophoretic units in which the recording electrode protruded by 50 pm and was fixed to the iontophoretic unit by Superepoxy (Engberg et al., 1979a). The central recording electrode was filled with 2% agar m 2.5 M KC1 (resistances of 4--8 M~t measured in 0.9% saline) and graphite screened (Engberg et al., 1975). The lontophoretic electrodes (tip diameter 5--9 pm) contained the following drugs: L-monosodium glutamate (1 M, pH 8.0-8.1), DL-monosodium homocysteate (0.3 M, pH 7.9--8.1, DLH), N-methyl-Dsodium-aspartate (0.2 M, pH 8, NMDA), sodium kainate (0.02 M, pH 8),

Fig. 1. S c h e m a t i c d r a w i n g o f t h e m i c r o m a n i p u l a t o r s a n d r e c o r d i n g s y s t e m . M i c r o m a n i p u l a t o r 1 was fixed t o m i c r o m a n i p u l a t o r 2 b y an a l u m i n i u m block. T h e w h o l e s y s t e m was a t t a c h e d to t h e arc of a T r a n s v e r t e x s t a n d (to t h e right). T h e m u l t i b a r r e l l e d e l e c t r o d e used for m i c r o i o n t o p h o r e s i s was held in a n a d j u s t a b l e a l u m i n i u m c l a m p fixed t o microm a n i p u l a t o r 2. T h e rigid vertical pillar o f this h o l d e r also supported the head stage o f a m p l i f i e r B. T h e casings o f t h e a m p h f i e r h e a d stages were drived at ×1 a m p l i f i c a t i o n ( b a n k w i d t h 10 MHz). T h e g r a p h i t e screen of t h e c e n t r a l m i c r o e l e c t r o d e could be conn e c t e d to the casing o f e i t h e r a m p l i f i e r via t h e reed relay R a n d 1 0 0 n F c a p a c i t o r s w h i c h p r e v e n t e d t h e s t a n d i n g voltage o f t h e casing (Eide, 1 9 6 8 ) b e i n g t r a n s f e r r e d t o t h e elect r o d e screen. T h e i n p u t o f a m p l i f i e r B was c o n n e c t e d t o o n e barrel o f t h e i o n t o p h o r e t i c p i p e t t e s filled w i t h 1 M NaCl e i t h e r d i r e c t l y or via a 4.7 n F capacitor.

207

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208 L-noradrenaline HC1 (0.2 M pH 6, NA), dopamme HC1 (0.25 M, ptf 5, DA/, and NaCl (1 M). The drugs were ejected by current balanced iontophoresls from barrels which were placed diametrmally opposite across the, lontophoretic unit. It was only necessary to apply a retaining voltage to th¢ ~ kainate solution. The 'return lead' from the microiontophoretlc current generator was connected to a Ag/AgC1 electrode which was buried in the back muscles. This was separate from the recording systems' reference electrode. The central recording electrode was connected to the input of a high input impedence amplifier (Eide, 1968) via a Ag/AgC1 half cell. Potential recordings were also made from one of the barrels (1 M NaC1) of the mntophoretlc unit coupled to the input of a similar amplifier (Fig. 1). To minimize the capacitive coupling between the two recording electrodes and to earth, the screen of the central electrode was driven at ×1 by one or other of the amplifiers. The screen connection was routed by a reed relay (Fig. 1). The outputs of the two amplifiers were fed to the two sides of a differential amplifier, whose o u t p u t was in turn displayed on a low fidehty fiat bed recorder. Thus three DC recordings were available: the mtracellular potential; the extracellular potential immediately outside the impaled neurone; and the differential of these two potentials, which is equivalent to the transmembrane potential (ETM)at the site of drug apphcation (Nelson and Frank, 1963). The intracellular (EM) and extracellular potentials (referred to an indifferent Ag/AgC1 electrode buried in the back muscles) were displayed on a high fidelity ink jet recorder (Mingograph EMT 80). The intracellular and transmembrane potentials were also displayed on a slow speed recorder (Servogor 220, BBC or Servograph REC 51, Radiometer). RESULTS During ejection of DLH and glutamate m the vminity of a m o t o n e u r o n e we noted that the central recording electrode became negative with respect to the reference electrode (Fig. 2). We call this potential change the focal potential (FP). For short applications (up to 40 sec) of excitatory amino acids, the time course of the FP was similar to that of the intracellular response of a m o t o n e u r o n e recorded subsequently or simultaneously. Moreover, the simultaneously recorded ventral root field (VRF; see Engberg et al., 1979b) recovered with a similar time course to the decline of the FP following DLH and NMDA application. FPs of up to --50 mV were seen. There was no direct correlation between the magnitude of the FP and the mtracellularly recorded drug response size. More intense lontophoretic ejections produced larger FPs. If repeated doses of the excitatory amino acids were applied at constant intervals, it was noted that the size of the FP increased to a maximum and then remained constant (Fig. 3). However, if the pause between drug applications was increased, the size of the response declined. With longer lasting amino acid applications the half time of recovery of the

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~'ig. 2. A c o m p a r i s o n o f t h e r e s p o n s e s t o a c u r r e n t b a l a n c e d m l c r o 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 D L H ( i n d m a t e d by the bar b e n e a t h t h e :races) o n the focal p o t e n t i a l ( F P ) r e c o r d e d e x t r a c e l l u l a r l y ( l o w e r r e c o r d ) a n d t h e intracellular p o t e n t i a l f r o m a n e a r b y m o t o n e u r o n e u p p e r r e c o r d ) . The F P was r e c o r d e d first w i t h t h e e l e c t r o d e tips s e p a r a t e d by 24 pro, and t h e n t h e central e l e c t r o d e was a d v a n c e d by L8/Am to p e n e t r a t e t h e m o t o n e u r o n e . T h e negative going F P began as s o o n as t h e D L H e j e c t i o n s t a r t e d and c o n t i n u e d to increase h r o u g h o u t t h e D L H application. In t h e u p p e r r e c o r d , D L H e v o k e d a rapid d e p o l a r i z a t i o n w h i c h led to r e p e t i t i v e firing as s h o w n by h e t h i c k black b a n d . T h e firing o f t h e m o t o n e u r o n e can also be seen o n the F P ( t h e black d o t s i n d m a t e the p e a k s o f the spikes) T h e mgative going d e f l e c t i o n s o n t h e E M trace are t h e a f t e r - h y p e r p o l a r i z a t i o n s o f a n t l d r o m i c a l l y e v o k e d APs. F o l l o w i n g the a c t i o n o f D L H h e m o t o n e u r o n e r e c o v e r e d to a slightly m o r e h y p e r p o l a r i z e d level t h a n in the p r e d r u g p e r i o d , a n d o n l y an IS spike was e v o k e d (SD pike block) as s h o w n b y t h e a b s e n c e o f AHPs. The t i m e c o u r s e o f r e c o v e r y o f t h e F P and t h e d e p o l a r i z a t i o n were similar.

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Fig. 3. The extracellular p o t e n t i a l changes in t h e ventral h o r n o f t h e spinal c o r d m r e s p o n s e t o l o n t o p h o r e t i c a l l y a p p l i e d C1-Na ÷ and Na+DLH -. The r e c o r d on t h e left s h o w s the r e s p o n s e t o t h e a p p l i c a t i o n o f CI-Na ÷ T h e r e was a small, i m m e d i a t e , negative ' c o u p h n g a r t i f a c t ' at t h e start and e n d o f e j e c t i o n with a s h g h t (1 m V ) f u r t h e r increase o f negativity during the e j e c t i o n . T h e p o l a r i t y o f the lont o p h o r e t l c c u r r e n t t h r o u g h the drug barrels was t h e n reversed t o eject Na+DLH - The small ' c o u p h n g a r t i f a c t ' was o f the o p p o s i t e polarity, b u t was f o l l o w e d by a large increase in negativzty (i.e. a FP). On r e p e a t i n g t h e D L H a p p l i c a t i o n a large FP was r e c o r d e d (right h a n d r e c o r d ) . S u b s e q u e n t a p p l i c a t i o n s ( n o t s h o w n ) p r o d u c e d FPs o f o n l y slightly greater a m p h t u d e . The smaller size o f t h e first FP evoked by DLH was partmlty due to t h e d e p l e t i o n o f DLH in the e l e c t r o d e tip during t h e passage o f Na ÷ s h o w n m the left h a n d r e c o r d .

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FP was slower than that of the simultaneously recorded membrane potential response from a motoneurone. Biogenic amines usually produced little change in the extracellular potential, but on a few occasions a small positive FP resulted. Initially, we dismissed these slow potential changes as artifacts of our electrodes and the technique (c.f. Kelly et al., 1969). However, the following evidence accumulated which suggests that the FPs are a function of the neuronal response to the agents administered: (a) The negative FP was seen only in areas of the ventral horn close to cells which responded to the amino acids. No FPs were seen in the white matter. Only small FPs were seen in the ventral horn where there was no evidence of the presence of motoneurones close to the electrode, despite the presence of a large VRF. (b) The negative FP could be evoked by all electrodes in which DLH produced motoneuronal depolarization. (c) Reversing the direction of current flow through the DLH barrel and the balancing NaC1 barrels produced no significant potential change (Fig. 3). (d) In the case of kainate, merely removing the retaining voltage was occasionally sufficient to evoke a very large FP, i.e. in the absence of an iontophoretic current. Although the time course of the FP during short drug applications was similar to that of the intracellularly recorded potential changes, the FP tended to continue to increase after the intracellular response had plateaued (Fig. 2).

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Fig. 4. FPs recorded during current balanced iontophoretic applications of NMDA and kainate. The FP evoked by NMDA developed and recovered more slowly than that evoked by an application of DLH (not shown). The application of a much smaller dose of kainate evoked a rapidly rising, large FP which remained when the ejecting current was stopped.

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Fig. 5. The r e d u c t i o n of DLH-evoked FPs by a c u r r e n t balanced l o n t o p h o r e t m a p p h c a t i o n of p e n t o b a r b i t o n e . Before the a p p h c a t l o n of p e n t o b a r b i t o n e , repeated doses of DLH (balanced l o n t o p h o r e t i c a p p h c a t i o n s of 132 nA for 20 sec) apphed at the times s h o w n by the short bars under the trace, evoked FPs of c o n s t a n t size. P e n t o b a r b l t o n e itself p r o d u c e d a small negative FP and decreased t h e a m p l i t u d e of the FP evoked by DLH. After the p e n t o b a r b i t o n e ejection, the DLH evoked FPs slowly increased m a m p l i t u d e and ultimately were larger t h a n during the pre-drug c o n t r o l period.

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213 Slow FPs were easily differentiated from the rapid DC shifts or 'coupling artifacts' which can result from imbalance of the ejecting currents. During excitatory amino acid application the extracellular electrode used to record the FP often recorded spike activity from a nearby neurone. The latent period from the time when the drug apphcation was started, until action potential firing was detected, was s~milar before and after penetration of a cell by the central recording electrode. The onset and recovery of the negative FP evoked by NMDA was much slower than that evoked by DLH or glutamate. The negative FP evoked by small currents of kainate was only partially reversible (Engberg et al., 1979b) and that to large currents, irreversible (Fig. 41. In extreme cases the negative FP could be recorded at distances of up to 1 mm from the tip of the lontophoretic electrode. Glycine produced no FP during the early part of ejection, and only after some time did a small negativity develop. The small FP occurred at the same time as the N~ wave of the ventral root field potential rapidly dechned (Engberg et al., 1979b). Since in these experiments the changes in the intracellular potential and the changes in the FP mirror each other, we believe that the FPs can be employed as a measure of cellular response in pharmacological experiments. For example, with intracellular potential recording, an iontophoretlc application of pentobarbitone was seen to reduce the depolarization evoked by DLH. Following the withdrawal of the recording electrode until it lay just outside the neurone, a similar application of pentobarbitone revermbly reduced the FP evoked by DLH (Fig. 5). Similar results were obtained m another experiment in which the mtracellular potential, the FP, and the algebraic sum of these potentials (the local 'transmembrane p o t e n t i a l ' ) w e r e recorded simultaneously. DISCUSSION During the iontophoresis of excitatory amino acids the development of FPs negative with respect to a distant reference electrode is probably related to cell depolarization. We consider, for the reasons given earlier, that we have excluded the possibility that these potentials are artifacts of the iontophoretic technique. Slow polarization changes of polarity corresponding to the direction of current passage can occur during unbalanced ejection of rather large iontophoretic current. These occur if a single 'non-polarizable' half cell (of small surface area) serves both as the reference electrode for the recording amplifiers and as the return pathway from the iontophoretic current generator. This does not mean, however, that artifacts of the same polarity as the FP do n o t occur. Indeed, we have often noted slowly developing depolarizations of motoneurones during the extracellular iontophoresis of chloride ions. However, even this response to chloride ions may also have a functional explanation.

214 When motoneurones are depolarized by excitatory amino acids an extracellular flow of current would be expected to flow from the dendrites (and initial segment) to the membrane site at which these agents act. This current flow would cause extracellular potential changes whose magnitude would be proportional to the current density at the tip of the electrode. This would be more intense in regions with a high density of a m m o acid sensitive membranes ('receptors'). We noted that the largest FPs were recorded when the electrode was close to motoneurones which fired action potentials in response to excitatory amino acid application. Since in all probability large iontophoretic applications of excitatory amino acids spread for distances as great as 300 pm (Herz et al., 1969) through the cord and excite more than one m o t o n e u r o n e the relationship of the recording electrode to an ensemble of excited motoneurones may be critical. At other sites where the neurones are smaller and less well orientated, the FP may be much smaller or in highly layered structures such as the hippocampus or cerebellum very much larger. Another possibility is that the FP is generated by a similar flow of current associated with the depolarization of glial cells as a consequence of an increase in extracellular potassium ion concentration (K+0) when nearby cells and cell processes are depolarized (see Somjen, 1975). Indeed, an increase of extracellular potassium ion activity (aK+0) has been measured in the frog spinal cord during application of glutamate and DLH (Sonnhof et al., 1978). In the spinal cord of cats the transmembrane potential of glial cells has been shown to be closely related to the aK*0 (Somjen 1975; K~i~ et al., 1975). When the potential of the intracellular electrode was related to a distant reference electrode, a clear depolarization of the glial cells was seen when the extracellular aK*0 was increased by an intense, repetitive stimulation of afferent pathways. Constanti and Galvan (1978) have also shown non-responsive cells in olfactory cortex slices (presumably glial cells) to depolarize in response to an increase in aK+0 and, moreover, to the application of excitatory amino acids. However, we have not observed depolarizations of non-responsive cells in the spinal cord in response to iontophoretic applications of excitatory amino acids. Our results are in agreement with those of Krnjevid and Schwartz (1967) who also showed non-responsive cells in the cat cerebral cortex to be unaffected by iontophoretic application of glutamate. Of course, it may not follow that the glial cells we studied were unaffected b y excitatory amino acids. The negative FP produced by the excitatory amino acids could change the potential of the extracellular environment of the glial cell b y an amount that was equal and opposite to the transmembrane depolarization of the glial cell. Somjen and Lothman (see Somjen, 1975) have simultaneously recorded changes in the E M of glial cells, the potential of their extracellular environment and aK÷0. In these experiments the change in transmembrane potential was closely correlated with the expected change in potential of a 'potassium electrode' for the correspond-

215 ing variation of aK÷0. However, when the aK÷0 increased, the increase of intracellular positivity always exceeded the increase in extracellular negativity (Fig. 1, Somjen, 1975). In our experiments, for no change in potential of the intraceUular electrode to have occurred, the changes of the FP and the transmembrane potential must have been equal and opposite. Thus it is unlikely that the glial cell alone generated the FP, but that neurones contributed to the extracellular current flow. It is unlikely that the firing of motoneurones alone could produce a sufficient rise in aK÷0 to evoke a FP as the tetanic antidromic stimulation of motoneurones has been shown not to cause a rise of aK÷0 in the ventral horn of the cat spinal cord (Somjen and Lothman, 1974; Lux and Liebl, 1974). Irrespective of whether the current producing the FPs is generated by glial cells or neurones, FPs can attain remarkably large amplitudes. A FP of 50 mV requires that a very large resistance must be present in the extracellular pathway between the recording electrode and the reference electrode. A hypothesis of h o w this resistance might arise has been developed by van Harreveld and Biersteker (1964) in their discussion of the generation of asphyxial potentials in the spinal cord of cat. It might be predicted that agents which hyperpolarize neurones and cause a passage of current from the soma to the dendrites would give rise to a positive FP. Indeed, NA occasionally produced small positive FPs. As the hyperpolarizing response to NA (when present) is small compared with the depolarization produced by the amino acids, the small action (or lack of action) of NA is n o t unexpected. Thus it appears that measuring FPs during extracellular studies of motoneuronal firing gives an indication of the membrane potential changes occurring. Moreover, smaller doses of these excitatory agents could be used, since the initial rise of the FP occurs before the appearance of action potential firing (Fig. 2). This would greatly increase the a m o u n t of information that could be gathered from extracellular studies while avoiding the difficulties associated with intracellular recording. The practical use of the FP is limited, however, in that it is probably generated b y more than one cell in the vicinity of the tip of the electrode and within range of the applied drug. The cells in this group are not necessarily homologous and may not have identical responses to the applied agonists (although we believe that we are working almost exclusively with motoneurones). The size and polarity of the FP will then reflect the net effect of the agonist in the area, rather than giving precise information on the state of polarization of 'identified' cells (see Bloom, 1974). Presumably, the FP may be likened to the classical 'ventral r o o t potential' recorded in spinal cord in vitro (Phillis, 1978) and thought to be related to the polarization of neurones which send their axons into the ventral root. During FPs the extraceUular environment becomes negative with respect to the reference electrode. Thus the transmembrane potential (ETM) of the m o t o n e u r o n e membrane abutting this region would be less than the potential difference between the intracellular and the distant reference electrode (EM). This raises the problem as to what is the most correct value for the

'effective' m e m b r a n e potential (the potential gradmnt integral which deter mines neuronal activity} during drug action: the potential of the recording electrode with respect to the distant reference electrode or to the electrode .lust outside the neurone? The determining factor is probably the extent to which the m o t o n e u r o n e m e m b r a n e 'sees ~ the FP. If large areas are involved, the FP will determine the effective m e m b r a n e potential. For example, kamate can produc~ ~ a large FP which ca~ be recorded m the grey m a t t e r up to 1 mm away from the site of application. Hence, for the cells lying within thin region the intracellular potential recorded with reference to a local extracellular electrode must be used to assess the "effective membrane potential', which, during large applications of kamate, is therefore about 0 inV. On the o t h e r hand, ff the extracellular potential change is locahzed to a small area o f the m e m b r a n e of a single m~urone, the 'effective m em brane potentml' of that particular neurone may ,rot be influenced by the FP. U n f o r tu n ately , it is almost impossible to give an accurate assessment of the 'effective me m br a ne potential'. During DLH depolarizations of up to 20 mV, the overshoot of the antidromlc spike was not significantly altered (if anything, it became more positive) although a FP of up to 40 mV could have been produced by the drug. Had the FP significantly altered the 'effective memb r an e potential', we should have expected to see the overshoot potential become m or e negative. This implies that the area of neuronal m e mb r an e which is supporting the AP is riot seeing very much of the FP. When measuring the current/voltage relationship of a m o t o n e u r o n e m the presence and absence of an iontophoretically applied drug, the developm e n t of a negative FP would complicate the interpretation of the curves. Should one plot EM, E,rM, or the "effective m e m b r a n e pot ent m l ' on the ordinate? Probably ETM should be em pl oye d, as it will more nearly reflect the potential difference 'seen" by the area of m em brane exposed t o the drug. This problem of the 'effective m e m br a ne pot ent m l ' is also of great importance when measuring the reversal potential of drug induced responses. If the i o n t o p h o r e s e d agent produces a large FP over a wide area of the cord, the true reversal potential might be considerably different from the potential related to the reference electrode. In the case of e x c i t a t o r y amino acids the true reversal potential would be substantmliy more positive than that measured. Thin might explain dmcrepancies between the reversal potentials of synaptic events and t hat of the putative transmitter (Curtm, 1970}. In conclusion, we consider that t he FP is a genuine potential related to cellular polarization and is n o t the result of electrode 'misbehaviour'. Even t h o u g h we are unable to say definitively whet her the FP is generated by changes in neuronal or glial cell polarization, we consider that the FP is closely related to changes in the m em br ane potential of nearby neurones. In "all th e experiments we have r e p o r t e d , the changes in FP have closely mirrored th e changes in intracellularly r e c or ded m e m b r a n e polarization. As FPs are more easily recorded than the resting m e m b r a n e potential, t h e y could be r ecor de d in conventional extracellular experiments through-

217

out the CNS. All that is reqmred is DC couphng and a slow pen recorder (whose characteristics are non critical). However, it is necessary to ensure very accurate current balancing to avoid 'coupling' DC artifacts, caused by a shght imbalance of the iontophoretlc current if flush-tipped recording and iontophoretic units are used. Alternatively, electrodes should be used where the recording electrode has a small separation from the mntophoretic barrels (see Crossman et al., 1974). The FP could serve as a useful measure of cell depolanzatmn during the screening of excitatory agents in the CNS. Furthermore, the FP may be used in pharmacological studies of the interaction of these agents and their putative inhib~tors. REFERENCES Bloom, F.E. (1974) To sprltz or not to sprltz the doubtful value of aimless lontcphoresis, Life Sci., 14: 1819--1834. ten Bruggengate, G., Lux, H.D. and Liebl, L. (1974) Possible relationships between extracellular potassium activity and presynaptm inhibition m the spinal cord of cat, Pflugers Arch. ges. Physiol., 349. 301--317 Constanti, A. and Galvan, M. (1978) Amino acid-evoked depolarization of electrmally inexcltable (neuroghal?) cells in the guinea pig olfactorv (',)rtex slice, Brain Res , 153 183--187. Crossman, A.R., Walker, R.J. and Woodruff, G.N (1974) Problems associated with lontophoretm studms in the caudate nucleus and substa:atla mgra, Neuropharmacology, 13 547--552. Curtis, D.R. (1970) Amino acid transmitters in the mammahan central nervous system. In Prec. IV Int. Congr. Pharmacol., Vol. 1, pp. 9--31 Elde, E. (1968) Input amplifier for intracellular potential and conductance measurements, Acta physiol, scand., 73 1--2A. Elde, E. and Kallstrdm, Y. (1968) Remotely controlled mlcro-mampulator for neurophysiologmal use, Acta physiol, scand., 73. 2A. Engberg, I , Flatman, J.A. and Lambert, J D C (19'75) A simple and cheap method of screening glass mlcroelectrodes, Brit. J. Pharmacol., 55 312--313P. Engberg, I., Flatman, J.A and Lambert, J D.C. (1978) The action of N-methyl-D-aspartlc and kainic acids on motoneurones with emphasis on conductance changes, Brlt J Pharmacol., 64 384--385P. Engberg, I., Flatman, J.A. and Lambert, J.D C. (1979a) The actions of excitatory amino acids on motoneurones in the feline spinal cord, J Physiol. (Lend.), 288 227--261. Engberg, I., Flatman, J.A and Lambert, J.D.C. (1979b) A comparison of extracellular and intracellular recording during extracellular mlcroiontophoresis, J. Neuroscl. M e t h , 1 219--233 van Harreveld, A. and Bmrstecker, P.A. (1964) Acute asphyxiation of the spinal cord and of other sections of the nervous system, Amer. J Physiol., 206 8--13. Herz, A., Zieglg~nsberger, W. and Farber, (;. (1969) Mlcroelectrophoretm studms concernmg the spread of glutamm acid and GABA m brain tissue, Exp. Brain Res., 9 221--235. Kelly, J.S., Krnjevld, K. and Somjen, G. (1969)Divalent cations and electrmal oroperties of cortical cells, J. Neurobiol., 2: 197--208. K ~ , N., Sykov~, E and Vykhck:~, L. (1975) Extracellular potassium changes in the spinal cord of the cat and their relatmn to slow potentials, active transport and impulse transmissmn, J. Physiol. (Lond.), 249 167--182.

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