Journal of Physiology (1988), 400. pp.

677-690 With 6 text-figures Printed in Great Britain

677

EXCITATORY AMINO ACIDS IN SYNAPTIC EXCITATION OF RAT STRIATAL NEURONES IN VITRO

BY E. CHERUBINI, P. L. HERRLING*, L. LANFUMEYt AND P. STANZIONEt From INSERM U.029, 123 Boulevard de Port Royal, 75014 Paris, France

(Received 21 May 1987)

SUMMARY

1. Intracellular recordings were made from rat striatal neurones in vitro. The cells had resting membrane potentials greater than -60 mV and action potentials greater than 70 mV with spike overshoot of 10-30 mV. 2. In the presence of bicuculline intrastriatal stimulation evoked an excitatory postsynaptic potential (EPSP). The relationship between EPSP amplitude and membrane potential was not linear. The EPSP decreased in amplitude and duration for values of membrane potential more negative than -80 mV and increased in amplitude and duration for values of membrane potential more positive than -50 mV. 3. The mean reversal potential for the EPSP recorded with electrodes filled with potassium methylsulphate was - 9-2 + 17 mV (mean + S.E.M.) in presence of bicuculline (30 utM). A similar reversal potential was obtained with CsCl-filled electrodes. 4. The endogenous broad-spectrum excitatory amino acid antagonist, kynurenic acid (100-500 JtM), reduced the EPSP in a dose-dependent way, maximally by 80 % at 500 /,M, but a residual depolarization remained even at high antagonist concentrations. This effect was associated sometimes with a membrane depolarization and an increase in input resistance. 5. In normal artificial cerebro-spinal fluid solution and at resting membrane potential the specific N-methyl-D-aspartate (NMDA) antagonist, (D,L)-2-amino-7phosphonoheptanoic acid ((D,L)-AP7), did not affect the EPSP amplitude. However, this antagonist partially reduced the EPSP amplitude when the membrane was depolarized beyond -50 mV by intracellular current injection. 6. The nicotinic cholinergic antagonist mecamylamine (10 /M) caused a partial (24+3 %) reduction of EPSP amplitude at resting potential in normal medium. However, in the cells where a reduction of EPSP amplitude was observed it was always accompanied by membrane depolarization (7-1 + 2-1 mV). (+)-Tubocurarine and hexamethonium were without effect at 10 /M.
* Present address: Sandoz Research Institute Berne Ltd, P.O. Box 2173, CH-3001 Berne, Switzerland. t Present address: INSERM U.288 Pitie Salpetriere, F-75013 Paris, France. t Present address: Clinica Neurologica, Universitit di Roma, Tor Vergata, Rome, Italy.

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7. When Mg2+ was removed from the bathing solution, the EPSP increased in amplitude (89+9-5%) and duration. In Mg2+-free medium at resting membrane potential (D,L)-AP7 (30 /tM) partially reduced EPSP amplitudes (59 + 25 %). 8. It is proposed that a major component of the EPSP evoked by intrastriatal stimulation is mediated by excitatory amino acids. At resting membrane potential and in normal medium only non-NMDA receptors seem to contribute to the synaptic depolarization, but at depolarized potentials and in AMg2+-free medium an NMDA receptor-mediated component of the EPSP can be demonstrated.
INTRODUCTION

Amino acids like glutamate and aspartate are thought to be important excitatory transmitters in many CNS neurones (Crunelli, Forda, Collingridge & Kelly, 1982; Collingridge, Kehl & McLennan, 1983; Ganong, Lanthorn & Cotman, 1983; Jahr & Jessel, 1985; Thomson, West & Lodge, 1985; Herrling, 1985; Thomson, 1986). They act probably through the activation of different receptor types: N-methyl-Daspartate (NMDA), quisqualate and kainate receptors (Watkins, 1986). The NMDA receptor can be blocked in a voltage-dependent way by Mg2+ (Nowak, Bregestovski, Ascher, Herbet & Prochiantz, 1984; Mayer, Westbrook & Guthrie, 1984). In earlier studies in situ where the pharmacology of the cortico-striatal EPSP was analysed, evidence was obtained that in the 'natural' environment and near resting membrane potential this synaptic potential was mediated in large part by non-NMDA receptors because none of its components could be affected by potent specific NMDA antagonists but only by the broad-spectrum excitatory amino acid receptor antagonist, kynurenic acid (Herrling, Morris & Salt, 1983; Herrling, 1985). Previous studies of slices have shown that intrastriatal stimulation evokes an EPSP followed by an IPSP (Kitai & Kita, 1984; Kita, Kita & Kitai, 1984; Kita, Kita & Kitai, 1985). The IPSP is mediated by y-amino butyric acid (GABA) (Lighthall & Kitai, 1983), but the nature of the transmitter(s) involved in the synaptic excitation is still unclear. Extracellular recordings of field potentials and intracellular recordings suggest that the synaptic excitation is mediated partly by the activation of a cholinergic nicotinic-type receptor (Misgeld & Bak, 1979; Misgeld, Weiler & Bak, 1980; Misgeld, Weiler & Cheong, 1982; Dodt & Misgeld, 1986) or by an excitatory amino acid receptor (Cordingley & Weight, 1986). The main aim of the present study was to determine the pharmacology of the locally evoked EPSP in striatal slices with respect to excitatory amino acids, to compare it to results obtained in situ on the cortically evoked EPSP in caudate neurones, and to determine if manipulations of the ionic medium or the membrane potential, which are easily possible only in slice preparations, would reveal an NMDA component to this intrastriatally evoked EPSP.
METHODS

Rat striatal slices were prepared following the technique previously described (Cherubini & Lanfumey, 1987). Slices (350 ,um thick) were cut and incubated at room temperature in artificial cerebrospinal fluid (ACSF) solution and bubbled with 95 % 02 and 5 % CO2. The slices were left to recover for 1 h, then were removed from the incubation bath and transferred to a recording chamber

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(Williams, Henderson & North, 1984). A completely submerged slice was laid on a nylon mesh and was superfused at a rate of 3 ml/min and at 36 C with artificial CSF of the following composition (mM): NaCl, 126; KCl, 3-5; NaH2PO4, 1-2; MgCl2, 1-3; CaCl2, 2; glucose, 11; NaHCO3, 25; gassed with 95 % 02 and 5% CO2 (pH = 7-2, 7 3). Two small pieces of 300 ,um o.d. platinum-iridium wires secured the slice on the mesh. Stimulation electrodes (twisted bipolar NiCr insulated wires, 50 ,um o.d.) were positioned into the striatium 300-500,um from the recording electrode. Stimulation parameters were: square pulses of 20-30 /us duration and 2-30 V. Intracellular recordings were made with electrodes filled with 2 M-potassium methylsulphate and in few cases with 2 M-CsCl (DC resistance 60-120 MQ). CsCl-filled electrodes were used to study the reversal potential of the EPSP evoked by intrastriatal stimulation. Bicuculline (30 ,JM) was usually added to the bathing solution in order to block the GABA-mediated IPSP that often follows the EPSP. Current was passed through the recording electrode by means of an Axoclamp-2 amplifier. Bridge balance was checked repeatedly during the course of the impalement. Capacitative transients with the electrode tip outside the neurone were reduced to a minimum by negative capacity compensation. Individual action potentials, EPSPs and electrotonic potentials were either digitized and stored on the floppy disk of a PDP 11/23 computer or displayed on a storage oscilloscope. Drugs were bath applied via a three-way tap system. Effects of different substances were observed within 20-30 s of the solution entering the bath and the equilibrium was apparently reached within 3-5 min with drugs interacting at membrane receptors. (+ )-Tubocurarine, mecamylamine and kynurenic acid were obtained from Sigma; (D,L)-2-amino-7-phosphonoheptanoic acid, (D,L)-AP7, was obtained from Sandoz; hexamethonium and bicuculline were obtained from Fluka: (D)-2-amino-5-phosphonopentanoic acid, (D)-AP5, from Cambridge Research Chemicals and y-(D)-glutamylaminomethyl sulphonate (GAMS) was a generous gift from J. C. Watkins, Bristol.

RESULTS

Cell characteristics Stable intracellular recordings were obtained from seventy striatal neurones for periods of up to 5 h. Although no intracellular staining of the neurones was attempted in this study, it is probable that most impaled cells were medium spiny neurones since other studies reported that the majority of intracellularly stained cells in the striatum were of this type (Bishop, Chang & Kitai, 1982; Lighthall & Kitai, 1983). Only cells satisfying the following criteria were included in this study: resting membrane potential greater than -60 mV (range from -65 to -74 mV), spike amplitude > 70 mV with spike overshoot of 10-30 mV, resting input resistance ranging from 31 to 60 MS2. The membrane time constant measured in ten neurones closely approximated a single exponential and was 10 + 0 8 ms (mean+ s.E.M.). In keeping with earlier studies in vivo and in vitro (Sugimori, Preston & Kitai, 1978; Kita et al. 1984), the voltage-current relationship of striatal neurones displayed a marked reduction in its slope for values of membrane potential more negative than -80 mV (anomalous rectification).

Ca2+ and voltage dependency of EPSPs Intrastriatal stimulation evoked depolarizing synaptic potentials that often triggered an action potential. The depolarizing potentials were blocked by superfusion of Co2+ (2 mM) or by a solution containing 10 mM-Mg2' and in which Ca21 was omitted. This depolarizing synaptic potential was similar to that already described in striatal slices and considered to be a monosynaptic EPSP (Kitai & Kita, 1984; Kita et al. 1984; Kita et al. 1985). We measured the reversal potential of the EPSP in six neurones with CsCl and with potassium methylsulphate-filled
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680 E. CHERUBINI AND OTHERS micropipettes. The reversals were around - 11 mX (N = 2 c?ells) in the former (Fig. 1) and -9-2 + 1-7 mV (IN = 4) in the latter experimental conditions. The relationship between the amplitude of the EPSP and membrane potential was not linear. During membrane hyperpolarization to values more negative than -80 mV. the amplitude of the EPSP was decreased and the decay time was shortened (Fig. 1). A deviation
A mV 33

30 .
0 .

154x_
-58 1
-90

15

15

E
0L en CL'

w
0

-o

-100

-50
-15
-

50= a
E
S

Membrane potential (mV)

A -lm
A

15 mV 16
ms

Fig. 1. Reversal potential of EPSPs evoked by intrastriatal stimulation. A, digitized chart records of EPSPs elicited by instrastriatal stimulation (triangle) at five different membrane potentials. The EPSP size increased with membrane hyperpolarization. It decreased and reversed its polarity with membrane depolarization. CsCl-filled recording electrode and bicuculline (30 uM) present throughout the experiment. B. the amplitudes of EPSPs shown in A are plotted against the membrane potential. The reversal occurs near -11 mV. Resting membrane potential was- 68 mV.

from linearity was also observed in the depolarizing direction for values of membrane potential more positive than -50 mV. W'henever action potentials were not triggered on top of the EPSPl. there was a slight increase in its amplitude and duration (Fig. 5). This increase was associated with an apparent increase in input resistance (Kita et al. 1984).

Effects of NMDA

the membrane potential Neurones in our slices did not display action potentials unless electrically or chemically stimulated. In three neurones with a resting membrane potential of around -65 mV NMDA perfused at a concentration of 10-20 /tM elicited a characteristic bursty firing pattern (Fig. 2A). In two other cells with a resting potential of around -80 mV NMDA at 20 ,UM only slightly depolarized the membrane. However, if the resting potential was decreased by intracellular current injection to about -60 mV the same NMDA concentration again elicited a bursty firing pattern (Fig. 2B).
on

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FAST EPSP IN STRIATAL NEURONES

A

681

NMDA(10gpM)

-65 mV kmV %..}. !jbM

D-AP5 (30 Mm)

NMDA (1 0 gM)
-65 mV

30 mV B 1 min

Control
-86 mV

NMDA (20 pM)

-86 mV

t_
_____ 4

j30 mV
1 min

10 s

Fig. 2. Effects on bath-applied NMDA on striatal neurones in vitro. A, superfusion of a striatal slice with 10 1SM-NMDA induced characteristic oscillations of the membrane potential which were accompanied by bursts of action potentials (upper trace). This excitatory effect of NMDA could be prevented if the slice was perfused with medium containing D-AP5 at 30 uM, a selective NMDA antagonist (Watkins, 1986), lower trace. The resting potential of this cell was around -65 mV. B, in a different neurone with a resting potential of around -86 mV an intracellular injection of a steady inward current (450 pA; on: upward arrow; off: downward arrow) induced a membrane depolarization with regularly spaced action potentials (upper trace). Superfusion of NMDA for a few minutes at resting potential caused only a small (2 mV) depolarization. Only when the membrane was depolarized by intracellular current injection did NMDA elicit its characteristic burst firing which stopped at the end of the intracellular current injection although NMDA was still present in the medium (lower trace). Action potential amplitudes were partly truncated by the chart recorder in this and some of the following

Figures.

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E. CHERUBINI AND OTHERS

Interactions of kynurenic acid and y- (D)-glutamylaminomnethyl sulphonate (GAJ1S) with EPSPs In order to examine the contribution of the excitatory amino acids to this EPSP we have tested the effects of kynurenic acid which is an endogenous broad-spectrum
A

Kynurenic acid (300 gM)

4 -85mV X 4K
500 ms 1 min
B

+8 min

+>j4 j44J 25mV

5 ms

Kynurenic acid (300 MM) -765 mV

A

110 mV

70-

50-

(5)

o
c

(5)

30-

(4)

CD10

(3)

100 200 300 Kynurenic acid concentration (#M)

Fig. 3. Depression of intrastriatally evoked EPSPs by kynurenic acid. A, continuous record of the membrane potential of a striatal neurone before, during and after superfusion of kynurenic acid. Upward deflections are action potentials evoked by intracellular injection of depolarizing current followed by EPSPs elicited by intrastriatal stimulation. Downward deflections are electrotonic potentials resulting from the repeated passage of a fixed outward current pulse (400 pA). Kynurenic acid caused a 2 mV depolarization and an increase of input resistance by 25 %. B, each panel represents seven superimposed EPSPs evoked by intrastriatal stimulation (triangle) of increasing strength. Stimulation frequency was 004 Hz. Kynurenic acid strongly depressed EPSPs at all stimulation intensities. Partial recovery was obtained 25 min after beginning of wash. In this cell kynurenic acid depolarized the membrane by 15 mV. However, the traces shown were obtained when the membrane potential was restored to its resting value by intracellular current injection. C, dose-dependent depression of the amplitude of intrastriatally evoked EPSPs by kynurenic acid. Each point represents the mean and S.E.M. of the samples (N in parentheses).

excitatory amino acid antagonist (Perkins & Stone, 1982) known to block NMDA-, quisqualate-, kainate- and L-glutamate-induced excitations in the striatum (Herrling, 1985) and to reduce EPSPs in several CNS structures (Ganong et al. 1983;

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Robinson, Anderson & Koerner, 1984; Herrling, 1985; Jahr & Jessel, 1985). Kynurenic acid was bath applied in concentrations of 100-500 /sM. In every neurone tested (N = 21), kynurenic acid caused a dose-dependent reduction of the EPSP amplitude (Fig. 3A-C). The depression caused by kynurenic acid began 1-2 min after the drug reached the tissue and achieved its maximal amplitude 3-5 min later. A full recovery was usually obtained after washing with a kynurenic acid-free solution. The
A

GAMS (l100 M) -68 mV

lL

GAMS (300 jM)

-70 mV

50 mV

+3 min

is
B

2 min

Control -68 mV

GAMS (100M)

Wash

GAMS (300 jM)

-70mV
A
A

_10mV 5 ms

Fig. 4. Dose-dependent depression of the intrastriatally evoked EPSP by GAMS. A, continuous record of a neurone before, during and after bath application of GAMS at 100 /tM (upper trace) and 300 /M (lower trace). Upward deflections are EPSPs evoked by intrastriatal stimulation. Downward deflections are electronic potentials resulting from repeated passage of a fixed current pulse (-200 pA in the upper trace, -400 pA in the lower). B, averages of four EPSPs shown in A. Upper trace at the lower dose, lower trace at the higher dose.

reduction of the EPSP was associated in ten neurones with no change of the membrane potential and in eleven cells with a depolarization of 3-16 mV. Measures on the EPSP during kynurenic acid application were always done after restoring the membrane potential to its control value by intracellular injection of current. Kynurenic acid also induced an increase in input resistance of maximally 40 % in thirteen neurones and no change in eight. In two of the latter kynurenic acid was applied at 500/M and caused a reduction of the EPSP amplitude by 75 and 80 %.

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E. CHERUBINI AND OTHERS Kynurenic acid reduced large-amplitude EPSPs evoked by high stimulus strength as well as small-amplitude EPSPs elicited by low stimulus strength (Fig. 3B). When kynurenic acid (150 ,UM) was applied in the presence of bicuculline (30 JIM), a similar reduction of the EPSP was observed (34 + 3 8 %, N = 4). A second broad-spectrum excitatory amino acid antagonist, y-(D)-glutamylaminomethyl sulphonate (GAMS), which is thought to be somewhat more selective for the kainate and quisqualate receptor (Davies & Watkins, 1985) was also tested for comparison. Its effects on locally evoked EPSPs in four cells at doses from 100 to 300 ,UM were indistinguishable from those of kynurenic acid described above: there was a 25-50 % reduction of EPSP amplitude, a small depolarization of around 5 mV and an occasional dose-independent increase of the input resistance of 10-50 % (see Fig. 4).
684
A

Control
mV -45
-50
-

(D, L)-AP7 (30 mM)

L
___

-60-70 -80

__ l

-90

10mV ,lA
5 ms

.o
0
I

F
L

70

s
50e
30

-110 -90 -70 -50 Membrane potential (mV)

Fig. 5. Effect of (D,L)-AP7 on the EPSP and input resistance. A, oscilloscope records of EPSPs evoked by intrastriatal stimulation (triangles) at different membrane potentials in control conditions and during superfusion of (D,L)-AP7. (D,L)-AP7 only reduced the amplitude of the EPSPs evoked at depolarized ( <-50 mV) potentials and as a consequence prevented them from reaching threshold for action potentials. B, relationship
between membrane potential and input resistance and during (0) application of (D,L)-AP7 (30 /uM).

(Rin) of the same neurone before (0)

Interaction of (D,L)-AP7 with EPSPs The specific NMDA receptor antagonist (D,L)-AP7 (Perkins, Collins & Stone, 1982) was also tested on EPSPs evoked by intrastriatal stimulation in slices kept in normal ACSF solution. At resting and more negative membrane potentials (D,L)-AP7 (30 JuM)

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had no measurable effect on EPSPs or input resistance. However, if the membrane potential was depolarized to values beyond -50 mV, (D,L)-AP7 (30,uM) reduced EPSP amplitudes by 31 + 4 % without affecting input resistance in three of four cells that displayed no action potentials on the EPSP (Fig. 5A).
Effects of Mg2+-free medium on EPSPs These observations, notably the voltage dependency and the antagonism of the EPSP by (D,L)-AP7, as well as the powerful effects of NMDA in striatal neurones
A

Control

0 Mg2+

Mg2+ + (D, L)-AP7

'I

-94 mV

B mV -60 -70--

-80 ~-~

-L.
A

&

-90

-10000 -110A

10mv 5 ms

Fig. 6. The amplitude of intrastriatally evoked EPSPs is enhanced in Mg2+-free medium. A, superimposed EPSPs evoked by intrastriatal stimulation (triangles) at a frequency of one per minute, in the presence (left) and absence (centre and right) of Mg2+. Perfusion with a Mg2+-free medium did not change the EPSP during the first eighteen stimuli. The arrow indicates EPSPs evoked during this period. However, starting from the 19th stimulation, there was an enhancement in the amplitude and duration of EPSPs. The right panel shows that (D,L)-AP7 (30 /LM) partly reverses the previous enhancement. The first EPSP obtained after the beginning of perfusion with medium containing (D,L)-AP7 is indicated by the arrow. Consecutive sweeps show gradually decreasing EPSP amplitudes and durations. B, voltage dependency of the EPSP shown in the presence (left) and in the absence of Mg2+ (centre). In Mg2+-free medium the EPSP increased in amplitude and duration with membrane hyperpolarization within a certain potential range. When (D,L)AP7 (30 uM) was added to the Mg2+-free solution (right) the EPSP decreased in amplitude and duration with membrane hyperpolarization as in control conditions.

(Herrling et al. 1983), raise the possibility that the EPSP is mediated, at least in part by a NMDA receptor. To further test this hypothesis, we performed experiments where the Mg21 concentration of the medium was modified because it was reported that the ion channel operated by NMDA is subject to a voltage-dependent blockade by Mg2+ ions (Nowak et al. 1984). First, we examined the effects of removing Mg2+ from the bathing solution on the EPSP. Within 20-30 min of superfusion with
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E. CHERUBINI AND OTHERS

Mg2+-free medium, the EPSP started to increase in amplitude and duration and reached a maximum after 40-60 min (Fig. 6A, middle panel). The mean percentage increase observed was 89-5 + 95 % (N = 5). A change in the voltage dependency of the EPSP described above was also observed: the EPSP increased in amplitude with membrane hyperpolarization, at least for values of membrane potential up to about - 100 mV. However, it still decreased in amplitude at even more negative values (Fig. 6B, middle panel).

Effects of (D,L)-AP7 on EPSPs in Mg2+-free medium

To determine if the increased amplitude and duration of the EPSP in Mg2+-free medium was due to the unmasking of NMDA-receptor mediated effects, we applied (D,L)-AP7 to augmented EPSPs. This NMDA antagonist at 30 #M reduced the amplitude and duration of EPSPs at resting potential by 59 + 2-5 % (N = 5; Fig. 6A, right panel). As before, membrane potential and input resistance were not affected by (D,L)-AP7. In presence of (D,L)-AP7 in Mg2+-free medium the voltage dependency of the EPSP lost its linearity and showed a marked decrease in amplitude and duration with increasing hyperpolarization (Fig. 6B, right panel). All these effects were reversible 10-20 min after restoring normal Mg2+ concentrations to the medium. In two experiments the slices were pre-incubated for longer time periods (5-7 h) in Mg2+-free medium. Under these conditions addition of (D,L)-AP7 (30 /LM) also reduced the amplitude and duration of the EPSP by 64 and 47 % without changing the membrane potential or input resistance of these neurones as with shorter perfusion times with Mg2+-free medium.
Interaction of cholinergic antagonists with EPSPs Because even at high concentration of kynurenic acid the EPSP was never entirely abolished and because of reports in the literature (Misgeld et al. 1982) that there may be a nicotinic cholinergic component to this synaptic potential, we tested mecamylamine, a nicotinic antagonist (Brown, 1980), on several cells at 1 and 10/tM. At concentrations of 1-10 /tM there was a variable reduction of EPSP amplitudes of 0-48 %, an equally variable increase of input resistance of 0-48 % and a variable depolarization of 0-18 mV (eight cells). However, (+ )-tubocurarine and hexamethonium, both at 10 /tM, were without effect on EPSPs (N = 3 in each case).

DISCUSSION

The present results provide evidence that the EPSPs elicited by local stimulation in neurones in striatal slices maintained in vitro are at least partly mediated by excitatory amino acids and that an NMDA component contributes to these synaptic depolarizations. The reversal potential of the EPSP observed here was around -10 mV measured with CsCl- and potassium methylsulphate-filled electrodes. This value is very similar to the one obtained by other workers in hippocampal slices where the synaptic involvement of excitatory amino acids has been well established (Crunelli et al. 1982). Neurones in our striatal slices respond to bath-applied NMDA with a characteristic
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bursty firing pattern accompanied by oscillations of the membrane potential which are similar to those observed in cat caudate neurones in situ after ionophoretic application of NMDA (Herrling et al. 1983). In the slice, as in situ, this effect can be antagonized by specific NMDA antagonists. These excitatory effects of bath-applied NMDA are voltage dependent (Fig. 2) similar to NMDA effects observed in spinal neurones in vitro (Mayer, Westbrook & Guthrie, 1984). A large part of the locally evoked EPSP can be inhibited by the broad-spectrum excitatory amino acid antagonist, kynurenic acid, in normal medium and at resting membrane potential. This agent is known to be specific for excitatory amino acid receptors, i.e. it has no effect on acetylcholine- or current-induced excitations (Perkins & Stone, 1982; Ganong et al. 1983; Herrling, 1985; Jahr & Jessel, 1985) and probably acts at a postsynaptic site (Cotman, Flatman, Ganong & Perkins, 1986). This sensitivity of the locally evoked EPSP to broad-spectrum excitatory amino acid antagonists was confirmed by a second agent (GAMS) thought to have a moderate selectivity for kainate receptors but which also interacts with quisqualate and NMDA receptors (Davies & Watkins, 1985; Salt, 1987). Under the same conditions the EPSP remained unaffected by a specific NMDA antagonist ((D,L)-AP7). This indicates that at resting membrane potential and in normal medium only quisqualate and/or kainate receptors contribute to the synaptic depolarization. Even relatively high concentrations of kynurenic acid (500 JM) and GAMS (300 /LM) do not completely abolish the locally evoked EPSP so that it could be assumed that this synaptic depolarization is evoked by more than one transmitter. Indeed, other authors have suggested a nicotinic component to this EPSP (Misgeld et al. 1979, 1980, 1982). Our own attempts with cholinergic antagonists were not conclusive because we observed some inhibitory effects of mecamylamine but none of tubocurarine or hexamethonium. These experiments were not extended as they were not within the main scope of this study. Our results where EPSPs were studied at different membrane potentials and in bathing solutions with or without Mg2' revealed properties compatible with the involvement of an NMDA agonist in this composite synaptic potential. A specific NMDA antagonist could reduce both amplitude and duration of the EPSPs if the membrane potential was depolarized beyond -50 mV or if Mg2+ was omitted from the medium. Under both conditions EPSP amplitude and duration were larger than controls in absence of the antagonist. Similar voltage and Mg2' dependency was demonstrated for NMDA responses in cultures of central neurones in voltage clamp experiments (Nowak et al. 1984; Mayer et al. 1984) and for cortical EPSPs thought to be mediated by NMDA receptors (Thomson et al. 1985; Thomson, 1986). The difference in the time course of the effects of kynurenic acid and NMDA (3-5 min to maximal effect) in contrast to the time required to see effects of perfusions with Mg2+-free Ringer solution (20-30 min) might be due to the fact that the former two agents interact with excitatory amino acid receptor sites at the cell membrane outer face while Mg2+ ions have to diffuse out of their binding site within the channel (Nowak et al. 1984). Furthermore, kynurenic acid and NMDA are compounds applied to the slice in the superfusion medium from which they can reach the receptors relatively quickly. However, to see the effect of removal of Mg2+ ions the slice must be perfused for a length of time with Mg2+-free medium until all endogenous Mg2+ ion
stores are significantly depleted.
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Although the origin of the synaptic terminals responsible for the intrastriatally evoked EPSP in the slice preparation cannot be determined, it can be assumed that terminals from cortical afferents are present in the striatal slices and might be excited by local electrical stimulation. The locally evoked EPSP as mentioned above is probably a multi-component synaptic potential some of which might be identical to those activated following cortical stimulation in caudate neurones in situ. This view is supported by our observations which in normal medium and at resting potential are identical to those obtained for the cortically evoked EPSP in situ (Herrling et al. 1983; Herrling, 1985) with respect to the effects of excitatory amino acid antagonists. Only broad-spectrum antagonists like kynurenic acid but not specific NMDA antagonists ((D,L)-AP7) affected the EPSP. There are several possible reasons for the origin of the synaptic NMDA component seen in the rat slice preparation but not in the cat caudate in situ: (i) since intrastriatal stimulation might activate afferents to striatal neurones that are not identical to those activated after cortical stimulation in situ, the NMDA component might be due to the actions of an NMDA agonist released from terminals activated after intrastriatal but not after cortical stimulation; (ii) an alternative possibility is that cortical stimulation in situ also results in the interaction of a transmitter with NMDA receptors but their characteristic effects could not be seen in the in situ studies (see above) because membrane potential and ionic composition of the extracellular fluid could not be easily manipulated. In the striatal slice, manipulation of either parameter was required in order to reveal the synaptic NMDA component. Species differences resulting in different excitatory amino acid receptor populations in striatal neurones in cats and rats are not likely as cat caudate neurones were shown to be distinctly responsive to NMDA agonists (Herrling et al. 1983; Herrling, 1985; Do, Herrling, Streit, Turski & Cuenod, 1986) and these responses were indistinguishable from those observed here. In conclusion our results suggest that the locally evoked EPSP in rat striatal slices is mediated at least partly by excitatory amino acid receptors of the non-NMDA type at resting membrane potential and in normal medium. Furthermore, a component involving activation of NMDA receptors could be revealed at membrane potentials more positive than -50 mV and in Mg2+-free medium. This is the first evidence of NMDA-mediated synaptic events in the mammalian striatum.
The authors wish to thank Drs Y. Ben-Ari and C. D. Hull for helpful discussions and reading the manuscript.
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BISHOP, G. A., CHANG, H. T. & KITAI, S. T. (1982). Morphological and physiological properties of neostriatal neurons: an intracellular horseradish peroxidase study in the rat. Neuroscience 7,

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