Brain Research, 102 (1976) 91-101 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

91

A N T A G O N I S M OF C O R T I C A L E X C I T A T I O N OF STRIATAL N E U R O N S BY G L U T A M I C ACID D I E T H Y L ESTER: EVIDENCE FOR G L U T A M I C ACID AS AN E X C I T A T O R Y T R A N S M I T T E R IN T H E RAT STRIATUM

H U G H J. SPENCER

Department of Psychobiology, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted July 9th, 1975)

SUMMARY

Rat striatal cells that were excited by cortical stimulation were found to respond to cortical stimulation with an average latency of 12 msec. Each response consisted of a variable number of spikes with, on the average, a less than 1:1 relationship between the stimulus and the number of spikes generated. Iontophoretic application of glutamic acid diethyl ester (GDEE), a substance reported to be a glutamate antagonist, at currents of + 50 to +125 nA in the vicinity of neurons excited by cortical stimulation, almost totally suppressed the excitation in 90 ~ of the cells, and this suppression was fully reversible. All cells were excited by glutamate. G D E E also suppressed neuronal excitation produced by iontophoretic aspartate, glutamate and DL-homocysteic acid. It is concluded from this study that an excitatory amino acid, either aspartic or glutamic, may function as the transmitter in the corticostriate projection.

INTRODUCTION

The presence of a corticostriate projection of considerable size, described in various animals4,5,12, 31, has aroused much attention and has been subjected to electrophysiological analysis by a number of workers1,9,19, 24. One dominant characteristic of this corticostriate input is that it appears to be excitatory, yet striatal cells which respond to cortical stimulation do not usually do so with spikes but with a complex EPSP-IPSP sequence as recorded intracellularlyl, 9. From anatomical data it appears that the majority of striatal cells are interneurons la,a3 whose axons are short and appear to terminate intrastriatally. These interneurons, which have extensive dendritic fields (approximately 500/~m in diameter), are reported to receive intrastriatal afferents on all segments of the dendrites. Extra-

92 striatal afferents, which appear to be primarily cortical, thalamic and nigral in origin, mostly terminate on the more proximal segments of the interneurons ~l- i,L This d ifterential distribution of the afferent terminals is associated with a differentiation m the nature of the synaptic boutons between those found on the proximal and distal dendritic segments l~j. Hull and Buchwald and coworkers 1,9 have described the nature of the synaptic response to afferents arising externally to the striatum as being excitatory, while intrastriatal afferents appear to be predominately inhibitory 1.~ 17,e,. Since the latter predominate ~i, striatal in terneurons, in the absence of any external excitatory drive, can be considered to be in a state of tonic inhibition. This explains the low probability of encountering spontaneously firing cells in the striatum, a phenomenon reported by numerous workers. However, despite this inhibitory input, some striatal cells will respond to cortical stimulation with spike discharges. Liles 19 has reported units in the cat striatum which could follow cortical stimulation rates of over 100 per second, while maintaining a l :1 relationship between the stimulus and evoked action potential and which appeared to be orthodromically driven. It appears from degeneration and Golgi impregnation studies that this corticostriate projection is monosynaptic and that the fibers of this pathway are of a very small diameter4, a,l~,~a,V'. Since there have been no reports of short-latency cortical responses following striatal stimulation and ~ince the fibers are very fine, the likelihood of being able to antidromically drive the corticostriate pathway to confirm its monosynaptic nature appears to be remote. Of the putative transmitter substances which have been described as occurring in the striatum, dopamine appears to be predominately inhibitorye,S,2'a: dopamineinduced excitations are generally slow in onset and decay, are not very consistent and are influenced by the nature and level of the anesthesia employed z,~n. Similarly, acetylcholine, which appears to be present in the striatum in high concentrations a,za, exerts both inhibitory and excitatory effects on striatal cells following iontophoretic applicatione,S,"2, e:~ and is even more sensitive to the anesthetic agent used. Sub-anesthetic doses of barbiturates block the ACh excitatory responses2, eg. Glutamic acid. a potent neuronal excitant, is present in high concentrations in the striatum, 10-12 umolelg (ref. 10), a level of concentration in the CNS second only to that found in the cortical ectosylvian gyrus, 12.4 #mole/g. One well described characteristic of the response to iontophoretically applied glutamate, or of related aspartic acid, is the rapidity of the onset of excitation and the rapidity of cessation of its effects once the glutamate ejecting current is turned off. in addition, the consistency with which these excitatory effects can be elicited, regardless of the anesthetic conditions used, is another characteristic of the responses to excitatory amino acids. Besides the ubiquity of its occurrence, which appears to dampen the enthusiasm of many investigators for considering glutamate as a transmitter candidate, one main problem in establishing the identity of action between glutamate and the unknown CNS excitatory transmitter has been the lack of an effective antagonist. L-Glutamic acid diethyl ester (GDEE) has been proposed as a possible antagonist of glutamate actions in the CNS. Haldeman and McLennan and coworkers 6,7,9~ have demonstrated

93 that G D E E can reversibly block synaptic activation of cat thalamic relay neurons, cuneate nucleus neurons and spinal cord interneurons. In addition, G D E E reversibly antagonizes the effects of iontophoretically applied glutamate, aspartate and its excitatory analogue, DL-homocysteic acid, and cysteic acid on these neurons without affecting the excitatory response to acetylcholine. The results reported in this study are an extension of an investigation aimed at establishing the pharmacological characteristics of striatal cells receiving cortical drive. It was of interest to determine whether glutamic acid might be the transmitter involved, especially since striatal cells are often exquisitely sensitive to iontophoretically applied glutamate 29. METHODS

Thirteen hooded rats of both sexes, 250-400 g weight, anesthetized with a penthrane air mixture following halothane induction, were used for these experiments. The gas mixture was supplied from a simple laboratory-constructed anesthesia machine which permitted the accurate control of the penthrane levels. A flow-through nose mask delivered the anesthetic gasses and eliminated the necessity for tracheostomy. The animals were maintained at stage 3 anesthesia. The skull was exposed with the animal in the stereotaxic frame and three or four 0.75 mm diameter burr holes drilled in the skull to accommodate the stimulating electrodes. The multibarrel microelectrode assembly was passed through a 2-mm diameter trephine hole located in the region of A 8.0, L 2.5 mm (ref. 18) and the exploratory recording tracks were confined to the body of the striatum. Miniature 6- or 7-barrel micropipette assemblies with a tip diameter of 4-8 ,um (ref. 29) were used for extracellular recording and drug application. The center recording barrel and one lateral current control barrel were filled with 2 M NaCI. The remaining barrels were filled with glutamate, as the monosodium salt (GLUT), Sigma (0.2 M, pH 7); aspartate, as the sodium salt (ASP) (0.2 M, pH 8); DL-homocysteic acid (DLH), Calbiochem (0.2 M, pH 7); L-glutamic acid diethyl ester (GDEE), Sigma (0.2 M, pH 4); and in several experiments, acetylcholine (ACh), Sigma (0.2 M, pH 3.5) was used. The electrodes were filled by centrifugation immediately before use. The details of the experimental and recording procedures have been given in previous communications 26-29. Briefly, the extracellularly recorded cell firing was amplified using a 100 Hz-10 kHz band-pass amplifier and fed into a monitor oscilloscope. The Y output of the oscilloscope passed via a window discriminator to an epochal ratemeter 26, the output voltage of which was displayed on a chart recorder (Fig. 3). The window discriminator output pulses were also fed into a Fabritek 1042 signal averager which was used to compute post-stimulus time-interval histograms (PSTH). Four cortical stimulating sites were used (Fig. 1); these were located at 1 : A 12-12.5, L 1-1.5; 2: A 10, L 2.0; 3: A 4.5-5.5, L 2-2.5; 4: A 0.5, L 2.5, according to the coordinates of K6nig and Klippel's atlas 18.

94

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Fig. 1. Diagrammatic dorsal view of the rat brain indicating the position of the various extrapyramidal structures, the location of the cortical stimulating sites (stars) and the recording site (shaded circle). The axes are those used in K6nig and Klippel's atlasTM, and are in millimeters. Abbreviations: cp, caudate-putamen (corpus striatum); ha, nucleus accumbens ; gp, globus pallidus; sn, substantia nigra ; amn, nucleus antero-medialis (thalamus); adn, nucleus antero-dorsalis (thalamus).

Cortical stimulation was delivered via parallel bipolar 36 g stainless steel stimulating electrodes which had a tip separation of 0.5 ram, the electrodes projecting through the dura for approximately 0.5 mm. Stimuli were delivered from RF isolated constant current stimulators which, in turn, were driven from a laboratory-constructed stimulator slaved to the Fabritek 1042 operating in the PSTH mode. Stimuli consisted of single 0.3 msec pulses, 2-6 mA intensity, which were above threshold for eliciting the responses. These were delivered at intervals ranging from 1 to 10 per second depending on the unit under scrutiny, when performing PSTH analyses. Units were revealed by advancing the microelectrode assembly slowly through the striatum while stimulating the cortical sites either sequentially (1-2-3-4) or singly. Once a unit was encountered which responded consistently to cortical stimulation, the following procedure was normally followed. A control PSTH (64-256 sweeps, depending on the excitability of the unit under investigation) was made of the response to stimulation of the most effective cortical site. During this control PSTH a cationic current equal to the G D E E ejecting current ( + 5 0 to + 125 nA) was passed from the lateral NaCI barrel. Next G D E E was applied with the same current and after an initial period of 3-5 min had elapsed a second PSTH was made, G D E E being ejected during this PSTH. After a recovery period of up to 10 min following the G D E E application, a 'recovery' PSTH was made in the same manner as the control. All PSTHs of each series contained the same number of sweeps. Following the PSTH series the responses of the unit to applied glutamate and other substances were tested and the efficacy of G D E E in antagonizing these responses

95 was determined. The majority o f units could be held for the 1-1.5 h required to complete the test sequences. RESULTS

R e s p o n s e s to cortical stimulation While there appear to be no intra- or extracellular studies on the responses o f rat striatal cells to cortical stimulation comparable to those carried out by Liles on cats 19 and by Buchwald et al. 1, the nature o f the responses observed in this study appear to be essentially similar. Although the n u m b e r o f cells responding with excitation to two or more cortical inputs was not specifically determined, at least a third of the 38 neurons encountered appeared to do so. The n u m b e r o f spikes generated in response to the single stimulus pulse was quite variable, from 1 to 5 or more in contrast to Liles' findings 19. Also, the n u m b e r o f spikes was to some extent a function o f both the stimulus strength and repetition rate. The majority o f units responded on less than a 1:1 basis and the response ratios, while not specifically examined, tended to vary somewhat during the experiment. It is possible that the responses recorded in some cases m a y have been derived from more than one unit, and while there was some variability o f spike height, it was no more marked than that which has been observed during intracellular studies z0.

Cells were often encountered that fired irregularly in response to stimulation at first, but with continuing stimulation (at 3/sec) the firing would become less erratic and often the n u m b e r o f spikes per response would increase. Similar responses to intrastriatal stimulation have been reported by M a r c o et al. 2°. Some cells tested showed a distinct post-tetanic potentiation o f firing following a stimulus burst. Unfortunately for lack o f suitable recording apparatus, it was not possible to analyze these characteristics in greater detail. Latencies as determined from PSTHs ranged from 4 to 20 msec (x -- 12 msec, s = 2.1) and varied according to the stimulation site (Table I). The latencies o f firing, TABLE I THE LATENCIES BETWEEN CORTICAL STIMULATION AND THE INITIATION OF STRIATAL C E L L FIRING, AS DETERMINED FROM P S T H

RECORDS

x, average latency; s, standard deviation and n, the number of cells responding to each of the stimulus sites. Site 1

2

3

4

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12.7 3.2 18

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Fig. 2. Post-stimulus time-interval histograms of the effects of G DEE on the responses of striatal cells to cortical stimulation of the sites listed at the left hand side. The first column is the control response, i -- +100 nA, the second column is the response during GDEE ejection (4 100 nA) and the third column is the recovery response, The instant of stimulation is indicated by the arrows in the top trace, and by the corresponding marks on the lower traces. The vertical marks above the arrow heads represent 10 counts, all histograms are to the same scale. The bottom two series of responses are from the same cell but in response to two different stimulation sites. Each trace, with the exception of the first, represent the accumulation of 64 responses; 2750 (top) 128 responses. The numbers at the side of the traces represent the depth of the cell below the cortical surface in microns, it can be seen that GDEE causes a marked depression in the intensity of the excitatory response to cortical stimulation.

as m a y be observed from Fig. 2, did n o t exhibit a high degree of constancy (approximately ~ 1-2 msec variability) a n d the latency of the later spikes were even less predictable, as is evident from the shape of the PSTH. Changes in stimulus parameters a n d the application o f G D E E or the current control did not alter the latency significantly.

Effect of GDEE on cortically evoked firing Since the Fabritek did not have the facility to integrate the PSTH, in order to c o m p a r e responses it was necessary to rate the m a g n i t u d e of the PSTH o n a 1 to 5 scale as is illustrated in the diagram in T a b l e H. In the majority of the 38 units tested, G D E E p r o d u c e d a powerful a n d reversible r e d u c t i o n in the n u m b e r a n d frequency of spikes generated in response to cortical s t i m u l a t i o n as is evidenced by the P S T H s (Fig. 2). In less than 10 ~] of the r e m a i n d e r of the cells tested were the n u m b e r of spikes

97

TABLE ll T H E E F F E C T OF

GDEE

A P P L I C A T I O N O N T H E I N T E N S I T Y OF T H E S T R I A T A L C E L L RESPONSES TO C O R T I C A L

STIMULATION

The PSTHs, which were generated in response to cortical stimulation, have been classified into 5 intensity classes, since the area of the histogram provides an index of the intensity of the neuronal response. Each successive class 0-5, illustrated at the top of the table) is approximately twice the area of the preceding class. The number of responses of each magnitude obtained during the control, GDEE and recovery periods, have been tabulated below. It is evident that there there is a pronounced reduction in the intensity of the neuronal response during the GDEE application as compared to the current controls (see text). A Chi-square analysis indicated that the difference between the control and GDEE responses was highly significant (P ,~ 0.01), while there was no significant difference between the control and recovery periods.

CLASS

-

1

2

3

4

CONTROL

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0

8

22

11

2

GDEE

-

20

13

7

2

1

0

4

17

12

3

RECOVERY-

5

produced unchanged by application o f G D E E . O f those units unaffected, most o f these responded with a latency o f less than 5 msec. In only one cell did G D E E produce a slight enhancement in the response to stimulation. All the cells tested were also excited by application of glutamate ions ( - - 1 0 to - - 5 0 hA). The retaining currents used to prevent leakage o f amino acids were approximately + 1 5 nA. Recovery from the effects o f G D E E was relatively rapid, full responsiveness returning in about 2-3 min. A Chi-square test o f the difference in the frequency o f the distribution o f the number o f responses o f each magnitude before, during and after G D E E application was made, using the magnitude criteria described above. Between the control and recovery periods there was no significant difference, while between the G D E E and either control period the difference was highly significant (P < 0.0l). Effect o f G D E E on amino acid-induced excitation G D E E was effective in antagonizing the excitation produced by iontophoretically applied glutamate, aspartate and DL-homocysteic acids (Fig. 3), and, in agreement with the findings o f H a l d e m a n et al. 6,7 and M c L e n n a n 21, required ejecting currents from between -+-50 to ÷ 125 n A to achieve this effect. Cationic control currents of the same magnitude either had no effect or enhanced the response slightly. There was little difference in the ability o f G D E E to block the responses o f the same magnitude to the 3 excitant amino acids tested on striatal cells (Fig. 3). However, on several cortical ceils tested, glutamate-evoked excitation was antagonized to a greater degree than that produced by oL-homocysteic acid which is in agreement with previous studies6,V,21.

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Fig. 3. Four epochal ratemeter records of striatal neuronal firing in response to iontophoretic application of excitatory amino acids, showing the depression of amino acid-induced firing by GDEE. Glutamate (G), DL-homocysteic acid (D), aspartate (A) and G D E E were applied for the durations and currents indicated below the traces. In cell A (3300/xm) G D E E caused complete inhibition of the amino acid-induced excitation, followed by rapid recovery. In cell B (2990/~m) GDEE caused an enhancement of a bursting type of spontaneous activity which was unrelated to the application of the amino acids. The current controls (i + 100 hA) can be seen to have no significant effect on the cell firing. In cell C, (3970/tin) G D E E inhibition of glutamate-induced firing without inhibition of spontaneous activity is illustrated. Cell D is a cortical cell (1480 #m) in which G D E E (4-40 nA) depresses glutamate excitation selectively.

S p o n t a n e o u s firing, w h e n p r e s e n t , was in m o s t cases u n a f f e c t e d by G D E E o r the c u r r e n t c o n t r o l , e v e n w i t h e j e c t i o n c u r r e n t s o f + 125 h A . N o n e o f the cells tested w e r e sufficiently s t r o n g l y o r c o n s i s t e n t l y excited by A C h to p e r m i t t h e d e t e r m i n a t i o n o f the effects o f G D E E

oll A C h e x c i t a t i o n .

99 DISCUSSION The degenerating fiber tracts which can be demonstrated terminating in the striatum following cortical lesions are comprised of fine poorly myelinated axons 3z, the majority being less than 1 # m in diameter. Such fibers are generally considered to have conduction velocities in the range of 0.5 to 1 m/sec (ref. 30). Assuming that in the rat the caudatopetal fibers course ipsilaterally in the callosum and then enter the striatum via the internal capsule bundles, as would appear to be the case from Webster's studies31, 3z, then an approximate estimate of the path length from sites 2 or 3 to the recording locus would be 8 ram. Assuming a monosynaptic pathway ~1, a latency of 12 msec would give a conduction velocity of 0.8 m/sec, a figure that agrees fairly well with previous studies 19. The observed variability of the latency of these spike responses is further evidence for the existence of these fine fiber pathways. The fine diameter of these axons makes them very difficult to stimulate antidromically and the lack of striatal-evoked cortical responses reported in the literature confirms this. The assumption that the corticostriate pathway terminates monosynaptically on striatal cells derives from both histological and neurophysiological evidence9,16,19, 20, although the very long latency responses ( ~ 25 msec) might be mediated via a corticothalamic-striate pathway. Since G D E E blocks both the synaptically induced and glutamate-induced excitation of striatal cells, this would suggest that an excitatory amino acid, such as glutamic acid, may be involved as the excitatory transmitter in the corticostriatal pathway. However, as has been repeatedly pointed out, there are many dangers inherent in the use of antagonists to evaluate possible neurotransmitter candidates. Nonspecificity of effect, local anesthetic actions, intrinsic actions and non-reversibility of action, among others, tend to obscure the desired effect. G D E E appears to have few drawbacks as a glutamic acid antagonist; while it is neither highly specific or potent in the striatum and in the cortex, it appears to preferentially and reversibly antagonize glutamate-induced responses6,7, zl. G D E E does not appear to have a local anesthetic effect; in this study the spontaneous firing of the majority of cells was not affected nor was there a noticeable change in spike amplitude 8, even with ejection currents of 125 nA. Although G D E E has been found to cause a block of excitation of spinal mononeurons by increasing membrane conductance in a manner similar to GABA, the lack of change in spontaneous activity of striatal cells observed in this study suggests that this effect might be peculiar to the spinal cord. Future intracellular studies on striatal cells in conjunction with G D E E will be required to give an unequivocal answer. Yarborough (personal communication) has confirmed that G D E E applied to rat cortical cells antagonized amino acid-induced excitations without affecting AChinduced excitation. The data presented here is further evidence for the supposition that either glutamic or aspartic acid functions as excitatory transmitter in the CNS, and that one of these substances is probably the transmitter in the corticostriate pathway; although until a more selective antagonist becomes available, it will be difficult to determine which.

100 ACKNOWLEI)GEM[!N 1~, I w i s h Io t h a n k Drs. V. H a v l i c e k , K. R. H u g h e s a n d L. M. J o r d a n o f t h e U n i v e r sity o f M a n i t o b a , W i n n i p e g , f o r t h e i r s u p p o r t , a d v i c e a n d c r i t i c i s m .

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101 23 OLIVIER,A., PARENT,A., SIMARD,H., ANDPOIRER,L. J., Cholinesteratic striatopallidal and striatonigral efferents in the cat and monkey, Brain Research, 18 0970) 273-282. 24 ROCHA MIRANDA, C. D., Single unit analysis of cortex-caudate connections, Electroenceph. clin. Neurophysiol., 19 (1965) 237-247. 25 SPENCER, J. H., Programmable nanoampere constant current sources for iontophoresis, Med. Biol. Engng, 9 (1971) 683-702. 26 SPENCER,H. J., An epochal ratemeter for neurophysiological studies, Electroenceph. clin. Neurophysiol., 33 (1972) 228-231. 27 SPENCER,H. J., Integrated circuit animal heater control, Physiol. Behav., l0 (1973) 977-979. 28 SPENCER, H. J., A microlathe for constructing miniature multibarrel micropipettes for iontophoretic drug appliation, Experentia (Basel), 29 (1973) 1577-1579. 29 SPENCER, H. J., AND HAVLICEK, V., Alterations by anaesthetic agents of the responses of rat striatal cells to iontophoretically applied amphetamine, acetylcholine, noradrenaline and dopamine, Canad. J. Physiol. Pharmaeol., 52 (1974) 808-813. 30 WAXMAN, S. G., AND BENNETT, M. V. L., Relative conduction velocities of small myelinated and non-myelinated fibres in the central nervous system, Nature New Biol., 238 (1972) 217 219. 31 WEBSTER, K. E., The cortico-striatal projection in the cat, J. Anat. (Lond.), 99 (1965) 329-337. 32 WEBSTER, K. E., Cortico-striate interrelations in the albino rat, J. Anat. (Lond.), 95 (1961) 532544. 33 ZIEGLGg.NSBERGER,W., AND PULL, E. A., Intracellular investigations on the effect of micro-electrophoretically applied glutamate antagonists upon spinal neurones of the cat, Naunyn-Schmiedeberg's Arch. exp. path. Pharmak., 277, Suppl. R.89, (1973).

Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum.

Rat striatal cells that were excited by cortical stimulation were found to respond to cortical stimulation with an average latency of 12 msec. Each re...
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