amino acid receptor-mediated transmission at primary afferent

J. exp. Biol. 124, 239-258 (1986) 2 3 9Printed in Great Britain © The Company of Biologists Limited 1986

AMINO ACID RECEPTOR-MEDIATED TRANSMISSION ATPRIMARY AFFERENT SYNAPSES IN RAT SPINAL CORD

BYT. M. JESSELLCenter for Neurobiology and Behavior and Howard Hughes Medical Institute,

Columbia University College of Physicians and Surgeons, 722 W 168th St,New York, NY 10032, USA

K. YOSHIOKA

Department of Pharmacology, Nagoya University School of Medicine, Nagoya,

Japan

AND C. E. JAHR

Section of Molecular Neurobiology, Yale University School of Medicine, NewHaven, CT, USA

SUMMARYIntracellular recording techniques have been used to provide information on the

identity of excitatory transmitters released at synapses formed between dorsal rootganglion (DRG) and spinal cord neurones in two in vitro preparations. Explants ofembryonic rat DRG were added to dissociated cultures of embryonic dorsal hornneurones and synaptic potentials recorded intracellularly from dorsal horn neuronesafter DRG explant stimulation. More than 80% of dorsal horn neurones received atleast one fast, DRG-evoked, monosynaptic input. In the presence of high divalentcation concentrations (5mmolP' Ca2+, 3mmoll~' Mg2+) the acidic amino acidreceptor agonists, L-glutamate, kainate (KA) and quisqualate (QUIS) excited alldorsal horn neurones which received a monosynaptic DRG neurone input, whereasL-aspartate and ./V-methyl-D-aspartate (NMDA) had little or no action. 2-Amino-5-phosphonovalerate (APV), a selective NMDA receptor antagonist, was relativelyineffective at antagonizing DRG-evoked synaptic potentials and L-glutamate-evokedresponses. In contrast, kynurenate was found to be a potent antagonist of aminoacid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapsesexamined. The blockade of synaptic transmission by kynurenate appeared to resultfrom a postsynaptic action on dorsal horn neurones.

Intracellular recordings from motoneurones in new-born rat spinal cord were usedto study the sensitivity of the la excitatory postsynaptic potential (EPSP) toantagonists of excitatory amino acids. Superfusion of the spinal cord with APV didnot inhibit the la EPSP but did suppress later, polysynaptic components of theafferent-evoked response. Kynurenate was a potent and selective inhibitor of the laEPSP, acting via a postsynaptic mechanism. These findings indicate that L-glutamate, or a glutamate-like compound, but not L-aspartate, is likely to be thepredominant excitatory transmitter that mediates fast excitatory postsynapticpotentials at primary afferent synapses with both dorsal horn neurones andmotoneurones.

Key words: neurotransmitters, amino acids, spinal cord.

240 T. M. JESSELL, K. YOSHIOKA AND C. E. JAHR

INTRODUCTION

The transmission of sensory information at primary afferent synapses in the spinalcord involves the release of sensory transmitters that elicit excitatory postsynapticpotentials (EPSPs) in spinal neurones (Eccles, 1964). The most intensively studiedafferent synapse in the spinal cord is that between muscle spindle afferents (group laafferents) and tf-motoneurones (Burke & Rudomin, 1977; Redman, 1979). In themammalian spinal cord, the chemical nature of the monosynaptic la excitatorypostsynaptic potential (EPSP) has been well documented. The la EPSP is precededby a distinct synaptic delay (Brock, Coombs & Eccles, 1952) and reverses in polaritywhen the membrane potential is depolarized (Coombs, Eccles & Fatt, 1955; Engberg& Marshall, 1979; Finkel & Redman, 1983). Moreover, synaptic transmission iscompletely and reversibly blocked by removal of extracellular Ca2+ (Shapolvalov,Shiriaev & Tamarova, 1979).

Intracellular recording from neurones in the superficial dorsal horn of rat spinalcord slices maintained in vitro has demonstrated that activation of cutaneous afferentfibres also elicits fast EPSPs in dorsal horn neurones (Urban & Randic, 1984). Inaddition, an increase in the frequency of dorsal root stimulation evokes, in the samepostsynaptic neurones, slow depolarizing potentials that persist for seconds orminutes.

The identity of the excitatory neurotransmitters released at sensory synapsesformed by cutaneous or muscle afferents has not been established (Redman, 1979;Fagg & Foster, 1983; Salt & Hill, 1983a,b). L-glutamate has been proposed as atransmitter in primary afferents based on its distribution in the spinal cord and dorsalroots (Graham, Shank, Werman & Aprison, 1967). It is the only amino acid found inthe dorsal roots and dorsal columns in higher concentrations than in the ventral roots(Graham et al. 1967; Duggan & Johnston, 1970; Roberts, Keen & Mitchell, 1973).Biochemical studies have demonstrated the release of endogenous stores of L-glutamate from regions of the CNS containing primary afferent terminals (Roberts,1974; Takeuchi, Onodera & Kawagoe, 1983). Iontophoretic and pressure appli-cation of L-glutamate depolarizes the majority of mammalian spinal neurones in vivoand in vitro (Ransom, Bullock & Nelson, 1977; Watkins & Evans, 1981; Salt & Hill,1983a,6) with a reversal potential (Mayer & Westbrook, 1984) similar to that of theEPSPs evoked by stimulation of DRG neurones (Engberg & Marshall, 1979; Finkel& Redman, 1983; MacDonald, Pun, Neale & Nelson, 1983). Confirmation of therole of L-glutamate as an excitatory neurotransmitter at primary afferent terminalshas been hindered by the lack of specific L-glutamate receptor antagonists and bydifficulties in administering these compounds, in a controlled way, at identifiedprimary afferent synapses.

Some clarification of the role of L-glutamate in central synaptic transmission hasderived from pharmacological studies indicating the existence of distinct amino acidreceptor subtypes (Watkins & Evans, 1981; Foster & Fagg, 1984). Selective ligandsare available for only one receptor subtype; xV-methyl-D-aspartate (N'MDA) is anagonist and 2-amino-5-phosphonovalerate (APV) an antagonist at the NMDA

Sensory transmission in spinal cord 241

subclass of L-glutamate receptors (Davies, Francis, Jones & Watkins, 1981). TheNMDA receptor can be further distinguished by a voltage-dependent block of theNMDA-activated ion channel by Mg2"1" and other divalent cations (Ault et al. 1980;Mayer, Westbrook & Guthrie, 1984; Nowak et al. 1984). Two other L-glutamatereceptor subclasses have been proposed on the basis of the selectivity of the rigidstructural analogues of L-glutamate, quisqualate (QUIS) and kainate (KA) (Watkins& Evans, 1981; Foster & Fagg, 1984). QUIS and KA receptors are not activated byNMDA and are not sensitive to APV at concentrations that are sufficient to blockNMDA receptors. Moreover, the ion channel associated with QUIS and KAreceptors does not appear to be subject to voltage-dependent blockade by Mg2+. Atpresent, no structural or functional distinction between QUIS and KA receptors onspinal neurones has been established, since no antagonists exist that distinguishreadily between these receptor subclasses. y-D-Glutamylglycine (DGG) and cis-2,3-piperidine dicarboxylate (PDA) antagonize all L-glutamate-activated receptors whilehaving no antagonistic effect on responses to non-amino acid compounds (Watkins &Evans, 1981; Foster & Fagg, 1984).

At low agonist concentrations, and at membrane potentials near rest, L-glutamateitself has mixed agonist activity with about equal contributions of NMDA andQUIS/KA receptors to L-glutamate-activated currents (Mayer & Westbrook, 1984).The specificity of action of L-aspartate appears to vary (Davies et al. 1982) althoughin mouse and rat spinal neurones in culture L-aspartate exhibits reasonable selectivityfor NMDA receptors (Mayer & Westbrook, 1984; Jahr & Jessell, 1985).

To provide more direct information on the identity of sensory transmittersreleased by DRG neurones and to characterize the postsynaptic receptors thatmediate fast EPSPs at sensory synapses, we have examined the action of excitatoryamino acid receptor ligands at synapses formed between dorsal root ganglion anddorsal horn neurones in cell culture. In addition, intracellular recording frommotoneurones in in vitro preparations of spinal cord from new-born rats (Otsuka &Konishi, 1974) has been used to examine the possibility that L-glutamate mediatesthe fast EPSP produced by a defined class of primary afferents, the group la musclespindle afferents. The use of these in vitro preparations has permitted analysis of theeffects of known concentrations of excitatory amino acid antagonists and of thespecificities of the synaptic receptors mediating the action of the afferent transmitter.

SYNAPTIC TRANSMISSION BETWEEN DRG AND DORSAL HORN NEURONES INCELL CULTURE

Several groups have used dissociated cultures of DRG and spinal cord neurones toexamine synaptic transmission at DRG—spinal cord, spinal cord-spinal cord andspinal cord—DRG synapses (Ransom et al. 1977; Choi & Fischbach, 1981;MacDonald et al. 1983). We have found that one major difficulty in studyingprimary afferent transmission between DRG and dorsal horn neurones in dissociatedcell co-cultures is the low detectable incidence of synaptically coupled pairs ofneurones. To overcome this problem we have grown explants of DRG neurones in


co-culture with dissociated dorsal horn neurones. Simultaneous stimulation of manyDRG neurones increases dramatically the probability of recording from dorsal hornneurones with monosynaptic sensory input (Jahr & Jessell, 1985).

Intracellular recordings have been obtained from dorsal horn neurones thatreceived monosynaptic DRG input. In low concentrations of divalent cations(3mmoll~ Ca , O^mmoll"1 Mgf+), spontaneous postsynaptic potentials aredetectable in more than 95 % of dorsal horn neurones. Since no spontaneous synapticactivity or action potentials are detectable when recording from DRG neurones inthe same cultures, the spontaneous EPSPs probably reflect input from other dorsalhorn neurones. Electrical stimulation of DRG explants evokes EPSPs in a highproportion of dorsal horn neurones located near the ganglion explant.

Postsynaptic responses in recording medium containing 3 mmol P 1 Ca2+ and0-9 mmol P 1 Mg2"1", are mediated in part by polysynaptic circuits. Superfusion of cul-tures with medium containing high divalent cations (5 mmol I"1 Ca2+ and 3 mmol 1~Mg^+) increases the spike threshold of dorsal horn neurones (Frankenhauser &Hodgkin, 1957; Jahr & Jessell, 1985) and decreases or blocks all but the earliestmonophasic EPSP.

Several characteristics of the synaptic potentials elicited in dorsal horn neuronesby extracellular stimulation of DRG indicate that they are monosynaptic in origin.Under conditions in which the divalent cation concentration in the recordingmedium is increased to a level sufficient to block spontaneous synaptic input to dorsalhorn neurones (MacDonald et al. 1983), monosynaptic EPSPs generated in dorsalhorn neurones by DRG stimulation can be studied without contamination fromdorsal horn neuronal input. Evoked EPSPs follow repetitive DRG stimulation at10 Hz and are monophasic with a constant latency, providing additional evidencethat they are monosynaptic. The high proportion of dorsal horn neurones thatreceive DRG input under these recording conditions has therefore made it possibleto compare the pharmacology of monosynaptic EPSPs with that of the potentialsevoked by various excitatory transmitter candidates on the same dorsal hornneurones.

Examination of the chemosensitivity of cultured dorsal horn neurones hasindicated that excitatory amino acids have excitatory actions consistent with theirroles as fast sensory transmitters (Jahr & Jessell, 1985). The sensitivity to ex-citatory amino acids and related compounds was tested only in those dorsal hornneurones that received monosynaptic sensory input. The effects of excitatory aminoacids and their analogues on dorsal horn neurones were consistent. L-glutamate(10-20/imoir1), QUIS (1-10/imolP1) and KA (10-20jzmoir1) were all potentexciters (Fig. 1). KA and L-glutamate were approximately equipotent, whereasQUIS was 10—20 times more potent. These agonists caused rapid depolarizations ofup to 40 mV. The depolarizations were often suprathreshold. In contrast, NMDAand L-aspartate, at concentrations up to 200/imol 1~ , either had no effect or elicitedonly small depolarizations (1-5 mV) after repeated application (Fig. 1). Dorsal hornneurones that were insensitive to L-aspartate, however, did receive monosynapticinput from DRG neurones (Fig. 2).


Glu

NMDA Asp

10 mV

10s

NMDA

Fig. 1. Effects of acidic amino acids and structural analogues on dorsal horn neurones.(A) Quisqualate (QUIS; 0-ls, 5 / imoir 1 ) , kainate (KA; Is , 20/ imoir1) and L-glutamate (Glu; Is , 20/tfnoll"1) were applied to the dorsal horn neurone (at thearrowheads) by pressure ejection from a pipette positioned within 30 fan of the soma.Resting potential, — 60 mV. (B) Responses of a different dorsal horn neurone to L-glutamate (0-5s, 20/rnioH"1), N-methyl-DL-aspartate (NMDA; Is , 200/wnoir1) andL-aspartate (Asp; I s , 200/imoll"1). Both L-aspartate and N-methyl-DL-aspartatewere applied six times as indicated by the arrowheads. Resting potential, - 6 5 mV.(C) Responses of another dorsal horn neurone to pressure application of quisqualate( Is , 2 / imoir1 ) , L-glutamate (0-5s, 20/xmoir1) and TV-methyl-DL-aspartate ( Is ,200^moll~'; eight applications). Resting potential, — 63 mV. Drugs were ejected at6-9-17-3 kPa. (From Jahr & Jessell, 1985.)

To determine whether amino acids, or compounds acting at amino acid receptorson dorsal horn neurones, might be transmitters at sensory synapses, we examined theeffect of several antagonists of excitatory amino acid-evoked responses on the DRG-evoked monosynaptic EPSP recorded from dorsal horn neurones. The tryptophanmetabolite kynurenate (Elmslie & Yoshikami, 1983; Ganong, Lanthorn & Cotman,1983; Perkins & Stone, 1982) was found to be the most potent antagonist of both theEPSP and the depolarization evoked by pressure ejection of L-glutamate, QUIS orKA (Fig. 3). Kynurenate, at a concentration of lmmolF1, completely blockedmonosynaptic EPSPs and amino acid-evoked responses.* In contrast, neither2-amino-4-phosphonobutyrate (APB) nor L-glutamate diethylester at concentrationsup to 1 mmoll"1 significantly affected the DRG-evoked EPSP or the responses ofdorsal horn neurones to L-glutamate, KA or QUIS. APB is considered to exert


presynaptic inhibitory actions at amino acid-mediated synapses, although in theseculture experiments, no inhibitory action was observed. At the same concentration,the selective NMDA receptor antagonist, APV, slightly reduced the amplitude of theEPSP and of the L-glutamate-evoked depolarization. PDA and DGG reduced theamplitude of the EPSP and the L-glutamate-evoked depolarization, but with apotency less than half that of kynurenate. Other compounds related to kynurenate,quinolinate, kynurenine, kynurenamine, xanthurenate and picolinate, had neitheragonist nor antagonist activity.

Since kynurenate was the most potent antagonist at DRG-dorsal horn synapsesidentified in these studies, additional experiments were performed to determine itssite and mechanism of action. The reduction by kynurenate of EPSP amplitude andof amino acid-evoked depolarization was not associated with a change in the inputresistance of dorsal horn neurones or with changes in the threshold, amplitude orduration of the action potential recorded intracellularly from the cell bodies of DRGneurones (Fig. 3).

The blockade of amino acid-evoked depolarization of dorsal horn neurones clearlydemonstrates a postsynaptic site of action of kynurenate. It is possible, however,that kynurenate antagonizes EPSPs by blocking action potential propagation in theaxons of DRG neurones presynaptic to dorsal horn neurones. Graded stimulation ofDRG explants revealed discrete steps in the amplitude of the EPSP, suggesting thatseveral DRG neurones can form functional synapses with a single dorsal hornneurone (Fig. 4). In the presence of kynurenate the same number of discrete stepscould be evoked, although the amplitude of each incremental step was reduced to asimilar extent (Fig. 4). Moreover, recruitment of each step in the EPSP occurred at a

^ W

5mVitAiit 4 ,

Glu lOmV20 ms A sP

10s

Fig. 2. Monosynaptic sensory neurone input in the absence of L-aspartate sensitivity.(A) Oscilloscope traces of four superimposed EPSPs. (B) Chart record obtained from thesame neurone, showing the responses to pressure application of L-aspartate (Asp; Is ,200/imoll"1; six applications) and of L-glutamate (Glu; Is , 20/zmoir1). The fastupward deflections occurring at 5-s intends are the evoked EPSPs shown at a faster timebase in A. Resting potential, - 6 4 mV. (From Jahr & Jessell, 1985.)


stimulus strength identical to that required before addition of kynurenate. Thesefindings indicate that kynurenate does not block spike propagation in DRG axonsand are consistent with a postsynaptic site of inhibition of the DRG-evoked EPSP.

Control, wash

Kynurenate 0-5 mmol 1

5mV

l

15 mV

100 ms

Fig. 3. Effects of kynurenate on dorsal horn and DRG neurones. (A) Responses of adorsal horn neurone to a hyperpolarizing current pulse injected through the recordingelectrode and stimulation of a nearby DRG explant before, during and after superfusionof 0-5 mmolP 1 kynurenate. Sixteen responses were averaged in each condition and thensuperimposed. (B) Chart records obtained from the same neurone shown in A, duringwhich excitatory amino acids were applied by pressure ejection. The responses to L-glutamate (G; I s , 20/imoH"1), quisqualate (Q; 0-2s, 2/jmolF1) and kainate (K; I s ,20/imoll"1) were greatly attenuated by kynurenate (0-5 mmol I"1). Drugs were ejectedat 6-9 kPa. The fast deflections occurring at 5-s intervals are the evoked EPSPs andhyperpolarizing conductance pulses shown at a faster time base in A. Resting potential,— 65 mV. (C) Action potentials recorded from a DRG neurone before, during and aftersuperfusion of 0-6 mmol T 1 kynurenate. Sixteen action potentials were averaged in eachcondition and then superimposed. Action .potentials were evoked by stimulation of theexplant of which this neurone was a peripheral member. In this neurone, potentialsappeared to be generated by antidromic invasion, since there was a distinct delay from thestimulus artifact (stimulus duration was 200 ms) and since the action potential could be'fractionated' into an initial segment spike by hyperpolarizing the neurone (not shown).Resting potential, - 7 5 mV. (From Jahr & Jessell, 1985.)

5mV

Fig. 4. The effect of kynurenate on EPSPs evoked by multiple DRG inputs. (A),(B) and(C) are EPSPs recorded from a dorsal horn neurone before, during and after washout of0-5 mmol I"1 kynurenate, respectively. EPSPs were evoked by electrical stimulation of anearby DRG explant at three distinct stimulus strengths. A complete recovery fromkynurenate was not achieved. All records are unaveraged responses photographeddirectly from an oscilloscope. Resting potential, — 66mV. (From Jahr & Jessell, 1985.)


Additional information on the site and mechanisms of action of kynurenate wasobtained by examining its effect on synaptic depression evoked by paired presynapticstimuli (Jahr & Jessell, 1985). At stimulus intervals of 50-400 ms, the amplitude ofthe second EPSP was depressed to 50-70 % of that of the first EPSP in mediumcontaining SmmolP1 Ca2+ and SmmolP1 Mg2"1". When the Mg2+/Ca2+ ratio wasincreased or Ca2+ channel blockers such as Co2+ or Cd2+ were added to thesuperfusion medium, synaptic depression was either partly or entirely abolished. Inexperiments in which the first EPSP was substantially decreased, a potentiation ofthe second EPSP resulted. In contrast, when the first EPSP was decreased to about20% of its control amplitude by addition of O-Smmoll"1 kynurenate, the secondresponse was decreased to a similar extent. In fact, the addition of kynurenatesometimes resulted in an enhancement of the synaptic depression that may be due toa slight use-dependency of kynurenate blockade. Comparison of the steady-stateEPSP amplitudes evoked at 0-2 Hz and 10 Hz in the presence and absence ofkynurenate revealed that the antagonism was enhanced approximately 1-2-fold at thehigher frequency.

Although kynurenate antagonized the response of dorsal horn neurones to L-glutamate, QUIS and KA, it did not inhibit the response of dorsal horn neurones toother excitatory transmitter candidates. We have shown previously that ATP excitesa subpopulation of dorsal horn neurones by activating a membrane conductancesimilar to that evoked by L-glutamate (Jahr & Jessell, 1983). The reversal potential ofboth L-glutamate- (Mayer & Westbrook, 1984) and ATP-evoked responses on spinalneurones is near OmV. Superfusion of dorsal horn neurones with concentrations ofkynurenate that were sufficient to antagonize both the DRG-evoked EPSP and theresponse to L-glutamate had no effect on the response of the same neurones to ATP(Jahr & Jessell, 1985).

SYNAPTIC TRANSMISSION AT la AFFERENT SYNAPSES IN VITRO

Examination of the chemosensitivity of DRG-dorsal horn neurone synapses inculture has provided strong evidence for the role of QUIS/KA receptors inmediating fast excitatory synaptic potentials. Our studies in culture, however, havenot yet attempted to identify the subclasses of DRG and dorsal horn neuronesthat represent the pre- and postsynaptic elements of the synapses examinedphysiologically. Moreover, the use of dorsal horn neurones precludes any com-parison of transmission at cutaneous and muscle afferent synapses. To assess thediversity of amino acid-like compounds as primary afferent transmitters, we haveused in vitro preparations of new-born rat spinal cord (Otsuka & Konishi, 1974) totest whether a specific class of primary afferents, the group la afferents, releases L-glutamate, or a similar compound, as a transmitter mediating the fast la EPSPrecorded from motoneurones (Jahr & Yoshioka, 1986). Two procedures were used toisolate the la EPSP from contaminating polysynaptic potentials. The sciatic nerveand its branches were dissected to the muscles which they innervated, permitting theselective stimulation of individual muscle nerves and smaller intramuscular nerve


branches and thus dramatically restricting afferent input. This greatly decreasedinterneuronal activation and resulted in the partial isolation of the la EPSP. Inaddition, as in the culture experiments, high concentrations of divalent cations wereadded to the recording medium in order to increase spike threshold (Frankenhauser& Hodgkin, 1957; Jahr & Jessell, 1985) and block polysynaptic transmission.

Intracellular recordings were obtained from motoneurones with stable restingpotentials between —52 and — 83 mV. Motoneurones were identified by antidromicspike invasion evoked by stimulating the ventral roots, the sciatic nerve or its largerbranches (tibial, common peroneal) or individual muscle nerves. In many cases,stimuli that were subthreshold for antidromic activation resulted in depolarizationsof 0-5—1 mV which occurred with a latency of onset very close to or identical withthat of the antidromic spike. These short-latency depolarizations probably resultedfrom antidromic activation of electrically coupled motoneurones (Fulton, Miledi &Takahashi, 1980).

The pattern of convergence of la afferent input to identified motoneurones in thenew-born rat spinal cord was similar to that observed in the adult cat spinal cord(Eccles, Eccles & Lundberg, 1957). The largest la input was invariably fromhomonymous muscle nerves while heteronymous muscle nerve stimulation producedsmaller EPSPs or had no effect. Stimulation of antagonist muscle nerves eliciteddepolarizing postsynaptic potentials which reversed in polarity at membranepotentials only slightly more depolarized than the resting potential. In motoneuronesin in vitro preparations of new born rat spinal cord, the Cl~ reversal potential isslightly positive to the resting membrane potential and therefore IPSPs aredepolarizing (Fulton et al. 1980; Konishi, 1982; Takashashi, 1984). Collectively,these observations indicate that the synaptic circuitry and membrane properties ofneurones in the 4-10 day new-born rat spinal cord have developed sufficiently topermit analysis of the mechanisms underlying the la EPSP.

To test whether excitatory amino acids, or agonists of amino acid receptors, mightbe transmitters at la synapses, the effects of several compounds which antagonize theresponses of motoneurones evoked by excitatory amino acids on the monosynapticla EPSP were examined. In medium containing low levels of divalent cations,polysynaptic pathways remained intact (Figs 5, 6). The addition of 30-100//moll"1

APV suppressed later components of the EPSP (Fig. 6). In the presence of high(4mmoll~1 Ca2+, 8mmoll~l Mg2"1") concentrations of divalent cations, whichblocked later components of the EPSP, the same concentration of APV had no effecton the residual EPSP (Jahr & Yoshioka, 1986).

Kynurenate, PDA and DGG were effective antagonists of the la EPSP (Fig. 6).Kynurenate was the most potent of these antagonists. Large reductions in theamplitude of the EPSP evoked by stimulation of the dorsal root or muscle nerve wereproduced by kynurenate at 250-500 ^moll"1 and a complete block was produced atmillimolar concentrations (Fig. 6). At these concentrations, kynurenate had noeffect on motoneurone resting membrane potential or input resistance (Jahr &Yoshioka, 1986).


A Low divalents B High divalents C Low divalents

Fig. 5. EPSPs evoked in a posterior biceps-semitendinosus (PBST) motoneurone bystimulation of the PBST muscle nerve in different concentrations of external divalentcations. (A) Three superimposed records of EPSPs evoked in the presence ofl ^ m m o U " 1 Ca2+ and l -15mmoir ' Mg2+. (B) EPSPs evoked by identical stimuli asthose in A after switching to superfusate containing 4 mmol I"1 Ca2+ and 8 m m o i r 'Mg 2 ^ (C) EPSPs evoked after returning to 1-25 mmol I"1 Ca2+ and 1- IS mmol I"1 Mg2*.Resting potential, —63 mV. (From Jahr & Yoshioka, 1986.)

The site and mechanism of action of kynurenate was investigated by examining itseffect on the depression of the la EPSP produced by repetitive stimulation of thehomonymous or heteronymous muscle nerves (Curtis & Eccles, 1960). When stimuliwere paired at intervals from 40 ms to 1 s, a marked diminution of the second EPSPwas observed. Decreasing the first EPSP to about 30% of its control amplitude bythe addition of 0-5 mmol I"1 kynurenate resulted in a decrease in the second EPSP toa similar extent. In contrast, when the Ca2+/Mg2+ ratio was decreased, or Cd2+ orCo2+ was added in order to decrease presynaptic release of transmitter, the use-dependent depression was decreased. These results parallel similar studies in culture

A Control

B 30/anoll"1 APV

C Control

D 0-3 mmol 1 kynurenate

E lmmoll 'kynurenate

F 3 mmol I kynurenate5mV

40 ms

Fig. 6. Effects of 2-amino-5-phosphonovalerate (APV) and kynurenate on responsesevoked in an unidentified motoneurone by stimulation of dorsal root L5. (A) Foursuperimposed responses recorded in control superfusate. (B) Responses in the presenceof 30/anoll"1 APV. (C) Responses recorded after washing out APV. (D)-(F) Responsesrecorded in the presence of 0"3, 1 and 3 mmol I"1 kynurenate, respectively. All responsesrecorded in the presence of 1-25 mmol 1~' Ca2 and l-15mmoll~' Mg2"^. Restingpotential, — 73 mV. (From Jahr & Yoshioka, 1986.)

n

J


Control B 1 mmol I"1 kynurenate

lmV

u CARBGlu CARB Glu CARB

Fig. 7. Responses of an unidentified motoneurone to L-glutamate (Glu) and carbachol(CARB). (A) The superfusate was switched for 15s to saline to which 0-5mmoll~' L-glutamate had been added. After the membrane potential had returned to rest, thesuperfusate was switched to one containing 0 1 mmol I"1 carbachol for 15 s. (B) Anidentical experimental paradigm to that in A was performed in the presence of 1 mmol I"1

kynurenate. Superfusion rate was 3 ml min"1. Responses in A and B were recorded in thepresence of 6xlO~7moll~1 tetrodotoxin, 2 mmol I"1 Ca2+ and 4 mmol I"1 Mg2"1". Restingpotential, — 69 mV. (From Jahr & Yoshioka, 1986.)

(Jahr & Jessell, 1985) and provide evidence that kynurenate also antagonizes the laEPSP by a postsynaptic mechanism.

The specificity of action of kynurenate in neonatal rat spinal cord was determinedby examining its effect on the responses of motoneurones to excitatory amino acidsand non-amino acid excitants. In the presence of tetrodotoxin, kynurenate had noeffect on motoneurone responses to carbachol at concentrations which greatlyreduced the depolarization produced by L-glutamate (Fig. 7). The specificity ofkynurenate was also tested by examining its action on recurrent inhibitory pathways.To enhance disynaptic activation, we superfused the new-born rat cord with3 mmol I"1 Ca2+ and 0-5 mmol I"1 Mg2+. Under these ionic conditions, ventral rootstimulation elicited a depolarization in motoneurones which reversed into ahyperpolarization when the resting membrane potential was decreased below— 60 mV by d.c. injection (Fig. 8). The reversal indicated that this potential was dueto a conductance increase of an ion, probably CP, with an equilibrium potential justpositive to the resting potential (Fulton et al. 1980; Konishi, 1982; Takahashi,1984).

In the cat spinal cord, ventral root stimulation results in the activation of recurrentaxon collaterals of motoneurones which release acetylcholine onto inhibitory inter-neurones, the Renshaw cells (Eccles, Fatt & Koketsu, 1954; Curtis & Ryall, 1966).Renshaw cells, in turn, release an inhibitory neurotransmitter, probably glycine,which evokes an IPSP in motoneurones (Eccles et al. 1954). To establish that theinhibitory pathway in the new-born rat spinal cord was the analogue of the recurrentcollateral circuit in the cat, we tested its sensitivity to the glycine antagonist,strychnine and to the nicotinic antagonist dihydro-/3-erythroidine. The addition ofstrychnine greatly reduced the response to ventral root stimulation while having verylittle effect on the early depolarizing phase of the response to dorsal root stimulation.Strychnine did, however, suppress the late, inhibitory component of the response todorsal root stimulation. Dihydro-)3-erythroidine similarly attenuated the response to


ventral root stimulation but had no effect on the response to dorsal root stimulation.In contrast to the effects of strychnine and dihydro-/S-erythroidine, kynurenate hadno effect on the recurrent IPSP while greatly reducing the la EPSP evoked by musclenerve stimulation (Fig. 8) and the response to dorsal root stimulation. These resultssuggest that the response of motoneurones evoked by ventral root stimulation isanalogous to the recurrent IPSP in the cat spinal cord (Renshaw, 1941, 1946;Jankowska & Roberts, 1972; Walmsley & Tracey, 1981) and is kynurenate resistant.

DISCUSSION

Diversity of primary sensory transmitters

Our studies with amino acid agonists and antagonists provide strong evidence thatL-glutamate, or a compound with similar affinity for the synaptic receptor, is thetransmitter mediating fast EPSPs at the majority of sensory neurone synapsesexamined in vitro. The experiments performed in cell culture, in particular, permit adiscrimination between L-aspartate and L-glutamate as potential sensory transmittercandidates. The failure of NMDA and L-aspartate to excite dorsal horn neurones thatreceived monosynaptic EPSPs and exhibited L-glutamate sensitivity providesevidence that L-glutamate, but not L-aspartate, mediates the fast afferent EPSPs.The inclusion of high Mg^+ concentrations in all studies on synaptic transmission inculture, however, leaves open the possibility that L-aspartate released from DRG

Control Kynurenate

A D /"S-v. G J

- ^ , ^^^°5fei

Fig. 8. Effect of kynurenate on the responses of a posterior biceps-semitendinosus(PBST) motoneurone to ventral root and PBST muscle nerve stimulation. (A)-(C)Responses evoked in control conditions by stimulation of the ventral root at membranepotentials of —45, —60 and — 85 mV, respectively. (D)-(F) la EPSPs evoked in controlconditions by PBST muscle nerve stimulation at the same membrane potentials as inA-C. (G)-(I) Responses evoked by ventral root stimulation as in A-C in the presence ofl m m o l F 1 kynurenate. (J)-(L) Responses evoked by PBST muscle nerve stimulationas in D—F in the presence of lmmoll"1 kynurenate. All responses recorded in thepresence of Smmoll"1 Ca2+ and lmmoll"1 Mg2 +. Resting potential, —69mV. (FromJahr & Yoshioka, 1986.)


neurones could contribute to sensory transmission via an action at synaptic NMDAreceptors that were not detected under these recording conditions.

Biochemical studies demonstrating release of L-glutamate from the terminalregions of primary afferent fibres have provided additional evidence that L-glutamateis a transmitter released from sensory terminals (Roberts, 1974; Takeuchi et al.1983). Few other endogenous compounds with L-glutamate-like excitatory prop-erties have been identified in the CNS (Luini, Tal, Goldberg & Teichberg, 1984).The high density of L-glutamate receptor binding sites observed in the superficialdorsal horn of the spinal cord is also consistent with a physiological role forL-glutamate at sensory synapses (Greenamyre, Young & Penny, 1984).

From studies in culture, the proportion of DRG neurones that release L-glutamate-like compounds as primary afferent transmitters is not clear. It is possiblethat the maintenance of neurones in culture has in some way restricted analysis to asubpopulation of primary afferent synapses. Studies on new-born rat spinal cordpreparations, however, demonstrate the ability of kynurenate to antagonize both laEPSPs (Jahr & Yoshioka, 1986) and cutaneous input (K. Yoshioka, unpublished) todorsal horn neurones. Primary afferents conveying diverse sensory modalities there-fore appear to release L-glutamate-like compounds as primary sensory transmitters inthe mammalian spinal cord.

The amino acid antagonist sensitivity of afferent synaptic potentials elicited byactivation of defined classes of sensory neurones in vivo may provide additionalinformation about the universality of L-glutamate as a primary afferent transmitterin the spinal cord (Davies & Watkins, 1983). The broad-spectrum amino acidantagonist PDA has been reported to antagonize extracellularly recorded dorsal hornneuronal responses to noxious mechanical, but not to noxious thermal stimuli (Salt &Hill, 1983a,6; Peet, Leah & Curtis, 1983). Intracellular recording from dorsal hornneurones in isolated guinea pig spinal cord preparations has also indicated that somehigh-threshold, afferent-evoked synaptic potentials may be insensitive to amino acidantagonists (Schneider & Perl, 1985). These observations raise the possibility thatsome primary afferents may release non-amino acid fast excitatory transmitters.

From our studies on the chemosensitivity of spinal cord neurones to putativesensory transmitters, the only endogenous compounds examined that have post-synaptic actions consistent with fast excitatory transmitter roles are the acidic aminoacids and the nucleotide ATP (Jahr & Jessell, 1983). While results in culture and inthe new-born rat spinal cord preparation indicate that L-glutamate-like compoundsare prevalent sensory transmitters, the possibility that ATP may be a sensorytransmitter released from a small proportion of primary afferent fibres cannot beexcluded. ATP selectively excites dorsal horn neurones in cat spinal cord in vivo(Salt & Hill, 1983a,6) in particular, those that receive low threshold C fibre input(Fyffe&Perl, 1984).

Several of the neuropeptides present within subsets of cutaneous sensory neuronesmay also act as transmitters at primary afferent synapses. Of the peptides localized insensory ganglia, the role of substance P has been studied in greatest detail (Otsukaet al. 1982; Jessell, 1983). Intracellular recording from dorsal horn neurones in


rat spinal cord slices has demonstrated that the slow, synaptically mediateddepolarization that is elicited by high-frequency dorsal root stimulation can bemimicked by application of substance P and blocked by substance P antagonists(Konishi, Akagi, Yanagisawa & Otsuka, 1983; Urban & Randic, 1984). Substance Pand other peptides may therefore be released as synaptic transmitters mediating slowsynaptic potentials in the dorsal horn. The release of peptides from specificsubclasses of afferent fibres is likely to have important, but as yet undefined,physiological roles in regulating the excitability of subsets of dorsal horn neuronesthat receive synaptic input mediated by fast sensory transmitters.

Mechanism and specificity of kynurenate antagonism

The use of kynurenate has been important in establishing that L-glutamate, or acompound interacting with L-glutamate receptors, is a transmitter at sensory neuronesynapses examined in vitro. The blockade by kynurenate of L-glutamate sensitivity ofspinal cord neurones at concentrations that also reduce or abolish the DRG-evokedmonosynaptic EPSP is consistent with the idea that L-glutamate is a primary sensorytransmitter. The enhanced synaptic depression that was sometimes observed in thepresence of kynurenate may indicate that kynurenate has channel-blocking activity inaddition to its action as a competitive antagonist at amino acid receptors. The reasonfor the variability in enhanced synaptic depression is not known. It is possible thatdistinct populations of dorsal horn neurones express different classes of amino acidreceptors/channels and that these are antagonized by kynurenate by multiplemechanisms. Our results in culture and in the neonatal rat spinal cord, however,clearly indicate that blockade of synaptic transmission by kynurenate can occurindependently of enhancement of synaptic depression or change in EPSP timecourse.

Several lines of evidence indicate that kynurenate antagonism is restricted toexcitatory amino acid-mediated synaptic potentials. In cultures of dorsal hornneurones derived from embryonic rat spinal cord, kynurenate antagonizes the effectof excitatory amino acids without affecting the response to ATP, which depolarizesdorsal horn neurones by a mechanism similar to that of L-glutamate (Jahr & Jessell,1983, 1985). The response of motoneurones to L-glutamate is blocked byconcentrations of kynurenate that have no effect on the depolarization produced bycarbachol. Kynurenate also has no effect on the depolarization of the ventral rootevoked by carbachol, serotonin, noradrenaline, y-amino butyrate or substance P atconcentrations that blocked responses to L-glutamate, L-aspartate, QUIS, KA andNMDA (C. E. Jahr & K. Yoshioka, unpublished).

The synaptic specificity of kynurenate was established by comparing its action onthe la EPSP to that of the disynaptic recurrent inhibitory input to motoneurones. Inthe new-born rat, responses to glycine and the recurrent IPSP are depolarizing(Konishi, Saito & Otsuka, 1975; Evans, 1978; Fulton et al. 1980). Both strychnineand dihydro-/3-erythroidine antagonized the response to ventral root stimulationindicating the existence of a recurrent inhibitory pathway similar to that in the cat.The lack of effect of kynurenate on recurrent IPSPs at concentrations that greatly


suppress the la EPSP demonstrates: (i) selectivity of kynurenate antagonism atcentral excitatory and inhibitory synapses, (ii) selectivity between excitatory syn-apses in the spinal cord mediated by amino acid and non-amino acid neuro-transmitters and (iii) selectivity of kynurenate for postsynaptic amino acid receptors.

Postsynaptic receptors and transduction mechanisms at afferent synapses

The development of amino acid agonists and antagonists with enhanced specificityhas made it possible to distinguish synaptic events that result from the activation ofNMDA and KA/QA receptors. In our studies in vitro, the small degree of inhibitionof monosynaptic DRG—dorsal horn and la afferent EPSPs produced by the selectiveNMDA antagonist APV indicates that amino acid receptors involved in the in-itial transduction of sensory information transmission are predominantly of theKA/QUIS class. The persistence of DRG-evoked EPSPs at Mg2* concentrationsthat are likely to block the NMDA receptor/channel complex provides additionalevidence for the involvement KA/QUIS receptors in afferent synaptic transmission.Since Mg2+ blockade of the ion channel associated with NMDA receptor activation ishighly voltage dependent, analysis of the voltage sensitivity of synaptic currents atafferent synapses might provide an additional means of assessing the contribution ofNMDA receptors to synaptic transmission. Finkel & Redman (1983) observed littlevoltage dependence in the la afferent motoneurone EPSP in cat spinal cord. Thisresult is consistent with an interaction of the afferent transmitter with KA/QUISrather than NMDA receptors. However, the voltage dependence of synaptic currentsmay not be a sensitive indicator of low levels of channel blockade by Mg + and it isdifficult, therefore, to exclude a contribution of NMDA receptors to synapticcurrents evoked by la afferents.

Activation of NMDA receptors may contribute to transmission at some inter-neuronal synapses in spinal cord. While APV has no effect on the la EPSP itspecifically antagonizes later, presumably polysynaptic, components of the post-synaptic response evoked by dorsal root or sciatic nerve stimulation in the new-bornrat spinal cord. Kynurenate, PDA and DGG are antagonists of both the early andlate components of the dorsal root-evoked EPSP. Whether the ability of thesecompounds to antagonize the late component of the EPSP is due to blockade ofmono- or polysynaptic input to motoneurones is unclear. APV-sensitive synapticpotentials have been described in Xenopus embryo spinal cord (Dale & Roberts,1985) although their origin is still not established. Spontaneous synaptic currentsrecorded at synapses between spinal interneurones and identified chick moto-neurones in dissociated cell culture have been reported to exhibit some voltagesensitivity (O'Brien, 1985). Studies on spinal cord—spinal cord EPSPs in dissociatedcell culture, however, have not revealed an APV- or voltage-sensitive component(Nelson, Pun & Westbrook, 1986). APV-sensitive EPSPs have also been reported atinterneuronal synapses onto cerebral cortical and hippocampal neurones (Thomson,West & Lodge, 1985; Thomson, 1986; Hablitz & Langmoen, 1986). While thereis therefore no conclusive evidence for the involvement of NMDA receptors inexcitatory transmission at afferent primary synapses in the spinal cord, excitatory


amino acid transmission at many interneuronal synapses in the CNS may include anNMDA-receptor-mediated component.

There is preliminary information about the kinetics and unitary conductance ofchannels associated with NMDA and KA/QUIS receptors on spinal and other CNSneurones. Nowack & Ascher (1984) have reported that NMDA-activated channelson mesencephalic neurones have conductances in the range of 50 pS whereas KA-and QUIS-activated channels have extremely small conductances. Consistent withits mixed agonist activity, in cultured cerebellar neurones L-glutamate itself appearsto activate at least two distinct channels with discrete unitary conductances of lessthan 0-5 pS and 50pS (Cull-Candy & Ogden, 1985). In horizontal cells in goldfishretina, L-glutamate, QUIS and KA each induce a current with an elementalconductance of 2-3 pS (Ishida & Neyton, 1985).

Voltage-clamp and single-channel recordings from cultured spinal neurones haveprovided information about the changes in ion permeability that are associated withactivation of NMDA and KA/QUIS receptors. Channels associated with bothclasses of receptors are permeable to Na+ and K+, consistent with the reportedreversal potential for amino acid agonist-induced currents at or about 0 mV (Mayer &Westbrook, 1984). The divalent cation sensitivity of the NMDA receptor/channelcomplex has been examined by several groups. Single-channel analysis on excisedpatches of CNS neurones has revealed that channel blockade in the presence ofextracellular Mg2"^, Co2+, Ni2+ and Mn + is associated with rapid closures of theNMDA channel (Nowack & Ascher, 1985). The time that channels remain in theclosed state is increased with hyperpolarization and with increased extracellulardivalent ion concentration. The modification of single-channel kinetics by these ionsis not associated with any change in the single-channel conductance. In contrast,Ca2+ and Ba2+, at high concentrations, do not affect channel kinetics but decreasesingle-channel conductance (Nowack & Ascher, 1985). The reversal potential ofNMDA-induced currents is sensitive to extracellular Ca2+ concentration (Mayer &Westbrook, 1985) suggesting that the NMDA channel is permeable to Ca2+. Recentexperiments with the calcium indicator Arsenazo III have provided more directevidence that the inward currents activated by NMDA in voltage-clamped spinalneurones are accompanied by calcium influx and an increase in intracellular Ca2+

(MacDermott et al. 1986). KA appears to be considerably less effective than NMDAin inducing intracellular calcium transients in spinal neurones (MacDermott et al.1986).

Amino acids have also been reported to stimulate inositol phosphate turnover incultured CNS neurones (Sladeczek et al. 1985; Nicoletti, Iadarola, Wrobleski &Costa, 1985). The generation of intracellular inositol tris- and other polyphosphatesis likely to result in the mobilization of intracellular calcium stores (Berridge &Irvine, 1984). The potency of amino acid agonists in triggering inositol phosphateturnover, QUIS > L-glutamate S> NMDA = KA (Sladeczek et al. 1985), differsfrom their ability to induce calcium influx (MacDermott et al. 1986). The activa-tion of amino acid receptors on spinal neurones could therefore lead to increasesin intracellular calcium by distinct mechanisms: (i) the depolarization-induced


activation of voltage-dependent calcium channels; (ii) changing the calcium per-meability of NMDA channels and (iii) an inositol trisphosphate-mediated mobiliz-ation of intracellular calcium stores.

Substance P and other peptides released by primary sensory neurones are alsoknown to enhance inositol phosphate turnover (Mantyh et al. 1984) and to increaseintracellular calcium (Womack, Hanley & Jessell, 1985). Activation of amino acidand peptide receptors on spinal neurones may therefore result in common intra-cellular signals. A convergence of these signalling systems could represent onemechanism by which interactions between sensory transmitters integrate sensoryinformation at primary afferent synapses in the spinal cord.

This work was supported by grants from NINCDS, The McKnight Foundationand The Muscular Dystrophy Association. We are grateful to A. MacDermott forcritical comments and to C. L. Miller for preparation of the manuscript.

REFERENCES

AULT, B., EVANS, R. H., FRANCIS, A. A., OAKES, D. J. & WATKINS, J. C. (1980). Selective

depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinalcord preparations. J. Physiol, Land. 307, 413-428.

BERJUDGE, M. J. & IRVINE, R. F. (1984). Inositol trisphosphate, a novel second messenger incellular signal transduction. Nature, Land. 312, 315—319.

BROCK, L. G., COOMBS, J. S. & ECCLES, J. C. (1952). The recording of potentials frommotoneurons with an intracellular electrode. J . Physiol., Land. 117, 431-460.

BURKE, R. E. & RUDOMIN, P. (1977). Spinal neurons and synapses. In The Handbook ofPhysiology. The Nervous System, vol. 1 (ed. E. Kandel), pp. 877-944. Bethesda: AmericanPhysiological Society.

CHOI, D. W. & FISCHBACH, G. D. (1981). GABA-mediated synaptic potentials in chick spinal cordand sensory neurons. J. Neurophysiol. 45, 632-643.

COOMBS, J. S., ECCLES, J. C. & FATT, P. (1955). Excitatory synaptic action in motoneurons.J. Physiol., Land. 130, 374-395.

CULL-CANDY, S. G. & OGDEN, D. C. (1985). Ion channels activated by L-glutamate and GABA incultured cerebeHar neurons of the rat. Proc. R. Soc. Ser. B 224, 367-373.

CURTIS, D. R. & ECCLES, J. C. (1960). Synaptic action during and after repetitive stimulation.J. Physiol., Lond. ISO, 374-398.

CURTIS, D. R. & RYALL, R. W. (1966). The excitation of Renshaw cells by cholinomimetics. ExplBrain Res. 2, 49-65.

DALE, N. & ROBERTS, A. (1985). Dual component amino-acid mediated synaptic potentials:excitatory drive for swimming in Xenopus embryos. J. Physiol., Lond. 363, 35-59.

DAVIES, J., EVANS, R. H., JONES, A. W., SMITH, D. A. S. & WATKINS, J. C. (1982). Differential

activation and blockade of excitatory amino acid receptors in the mammalian and amphibiancentral nervous system. Comp. Biochem. Physiol. 72C, 211-224.

DAVIES, J., FRANCIS, A. A., JONES, A. W. & WATKINS, J. C. (1981). 2-Amino-phosphonovalerate

(2APV), a potent and selective antagonist of amino acid-induced and synaptic excitation.Neumsci. Letts 21, 77-81.

DAVIES, J. & WATKINS, J. C. (1983). Role of excitatory amino acid receptors in mono- andpolysynaptic excitation in the cat spinal cord. Expl Brain Res. 49, 280-290.

DUGGAN, A. W. & JOHNSTON, G. A. R. (1970). Glutamate and related amino acids in cat spinalroots, dorsal root ganglia and peripheral nerves._?. Neurochem. 17, 1205-1208.

ECCLES, J. C. (1964). The Physiology of Synapses. New York: Springer-Verlag.


ECCLES, J. C , ECCLES, R. M. & LUNDBERG, A. (1957). The convergence of monosynapticexcitatory afferents on to many different species of alpha motoneurons. J. Physiol., Land. 137,22-50.

ECCLES, J. C , FATT, P. & KOKETSU, K. (1954). Cholinergic and inhibitory synapses in a pathwayfrom motor-axon collaterals to motoneurons. J'. Physiol., Land. VZlb, 524—562.

ELMSLIE, K. S. & YOSHIKAMI, D. (1983). Effects of kynurenate on root potentials evoked bysynaptic activity and amino acids in the frog spinal cord. Soc. Neurosci. Abstr. 9, 263.

ENGBERG, I. & MARSHALL, K. C. (1979). Reversal potential for la excitatory post-synapticpotentials in spinal motoneurons of cats. Neurosci. 4, 1583-1591.

EVANS, R. H. (1978). The effects of amino acids and antagonists on the isolated hemisected spinalcord of the immature rat. Br.J. Pharmac. 62, 171-176.

FAGG, G. E. & FOSTER, A. C. (1983). Amino acid neurotransmitters and their pathways in themammalian central nervous system. Neurosci. 9, 701-719.

FINKEL, A. S. & REDMAN, S. J. (1983). The synaptic current evoked in cat spinal motoneurons byimpulses in single group la axons. J. Physiol., Land. 342, 615-632.

FOSTER, A. C. & FAGG, G. E. (1984). Acidic amino acid binding sites in mammalian neuronalmembranes: their characteristics and relationship to synaptic receptors. Brain Res. Rev. 7,103-164.

FRANKENHAUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical propertiesof squid axons. J. Physiol., Land. 137, 218-244.

FULTON, B. P., MILEDI, R. & TAKAHASHI, T. (1980). Electrical synapses between motoneurons inthe spinal cord of the newborn rat. Proc. R. Soc. Ser. B 208, 115-120.

FYFFE, R. & PERL, E. R. (1984). Selective excitation of a subpopulation of cat dorsal horn neuronsby ATP: Possible role as an excitatory sensory transmitter. Proc. natn. Acad. Sci. U.SA. 81,6890-6893.

GANONG, A. H., LANTHORN, T. H. & COTMAN, C. W. (1983). Kynurenic acid inhibits synapticand acidic amino acid-induced responses in the rat hippocampus and spinal cord. Brain Res. 273,170-174.

GRAHAM, L. T., SHANK, R. P., WERMAN, R. & APRISON, M. H. (1967). Distribution of somesynaptic transmitter suspects in cat spinal cord: glutamic acid, aspartic acid, gamma-aminobutyric acid, glycine and glutamine. J. Neurochem. 14, 465—472.

GREENAMYRE, J. T., YOUNG, A. B. & PENNY, J. B. (1984). Quantitative autoradiographicdistribution of L-[3H]glutamate-binding sites in rat central nervous system. J. Neurosci. 4,2133-2144.

HABLITZ, J. J. & LANGMOEN, I. A. (1986). N-Methyl-D-aspartate receptor antagonists reducesynaptic excitation in the hippocampus..7. Neurosci. 6, 102-106.

ISHIDA, A. T. & NEYTON, J. (1985). Quisqualate and L-glutamate inhibit retinal horizontal cellresponses to kainate. Proc. natn. Acad. Sci. U.SA. 82, 1837-1841.

JAHR, C. E. & JESSELL, T . M. (1983). ATP excites a subpopulation of rat dorsal horn neurons.Nature, Land. 304, 730-733.

JAHR, C. E. & JESSELL, T. M. (1985). Synaptic transmission between dorsal horn neuronsin culture: antagonism of monosynaptic EPSPs and glutamate excitation by kynurenate.J. Neumsci. 5, 2281-2289.

JAHR, C. E. & YOSHIOKA, K. (1986). la afferent excitation of motoneurons in the in vitro new-bornrat spinal cord is selectively antagonized by kynurenate. J. Physiol., Land. 370, 515-530.

JANKOWSKA, E. & ROBERTS, W. J. (1972). Synaptic actions of single interneurons mediatingreciprocal la inhibition of motoneurons..7. Physiol. 222, 623-642.

JESSELL, T. M. (1983). Substance P in the nervous system. In Handbook of Psychopharmacology(ed. L. L. Iversen, S. M. Snyder & S. D. Iversen), pp. 1-105. London: Plenum Press.

KONISHI, S. (1982). Electrophysiology of mammalian spinal cord and sympathetic ganglia in vitro.Adv. Pharmac. Ther. 2, 255-260.

KONISHI, S., AKAGI, H., YANAGISAWA, M. & OTSUKA, M. (1983). Enkephalinergic inhibition ofstore transmission in rat spinal cord. Neurosci. Letts (Suppl.) 13, S107.

KONISHI, S., SAJTO, K. & OTSUKA, M. (1975). Postsynaptic inhibition and glycine action inisolated spinal cord of newborn rats. Jap. J. Pharmac. (Suppl.) 25, 72-73P.

LUINI, A., TAL, X., GOLDBERG, O. & TEICHBERG, V. I. (1984). An evaluation of selected brainconstituents as putative excitatory neurotransmitters. Brain Res. 324, 271-277.


MACDERMOTT, A. B., MAYER, M. L., WESTBROOK, G. L., SMITH, S. J. & BARKER, J. L. (1986).

NMDA-receptor activation elevates cytoplasmic calcium in cultured spinal neurons. Biophys.Soc.Abstr. Vol. 365a.

MACDONALD, R. L., PUN, R. Y. K., NEALE, E. A. & NELSON, P. G. (1983). Synaptic interactionsbetween mammalian central neurons in cell culture. I. Reversal potential for excitatorypostsynaptic potentials. J . Neurophysiol. 49, 1428-1441.

MANTYH, P. W., PINNOCK, R. D., DOWNES, C. P., GOEDERT, M. & HUNT, S. P. (1984).

Correlation between inositol phospholipid hydrolysis and substance P receptors in rat CNS.Nature, Land. 309, 795-797.

MAYER, M. L. & WESTBROOK, G. L. (1984). Mixed agonist action of excitatory amino acids onmouse spinal cord neurons under voltage clamp. J. Physiol., Land. 354, 29-53.

MAYER, M. L. & WESTBROOK, G. (1985). Divalent cation permeability of N-methyl-D-aspartatechannels. Soc. Neurosci. Abstr. 11, 785.

MAYER, M. L., WESTBROOK, G. & GUTHRIE, P. B. (1984). Voltage-dependent block by Mg + + ofNMDA responses in spinal cord neurons. Nature, Land. 309, 261-263.

NELSON, P. G., PUN, R. Y. K. & WESTBROOK, G. L. (1986). Synaptic excitation in cultures ofmouse spinal cord neurons: receptor pharmacology and behaviour of synaptic currents.J. Physiol., Land, (in press).

NicoLETn, F., IADAROLA, M. I., WROBLESH, J. T . & COSTA, F. (1985). Excitatory amino acidreceptors are coupled with inositol phospholipid metabolism in the rat central nervous system.Soc. Neurosci. Abstr. 11, 822.

NOWACK, L. M. & ASCHER, P. (1984). N-methyl-D-aspartate, kainic and quisqualic acid evokedcurrents in mammalian central neurons. Soc. Neurosci. Abstr. 10, 23.

NOWACK, L. M. & ASCHER, P. (1985). Divalent cation effects on NMDA-activated channels can bedescribed as Mg-like or Ca-like. Soc. Neurosci. Abstr. 11, 953.

NOWAK, L., BREGESTOVSKI, P., ASCHER, P., HERBERT, A. & PROCHIANTZ, A. (1984). Magnesium

gates glutamate-activated channels in mouse central neurons. Nature, Land. 307, 462-465.O'BRIEN, R. (1985). Properties of identified motoneurons in culture. Ph.D. thesis, Harvard

University.OTSUKA, M. & KONISHI, S. (1974). Electrophysiology of mammalian spinal cord in vitro. Nature,

Land. 252, 733-734.OTSUKA, M., KONISHI, S., YANAGISAWA, M., TSUNOO, A. & AKAGI, H. (1982). Role of substance

P as a sensory transmitter in spinal cord and sympathetic ganglia, dba. Fdn Symp. 91, 13-34.PEET, M. J., LEAH, J. D. & CURTIS, D. R. (1983). Antagonists of synaptic and amino acid

excitation of neurons in the cat spinal cord. Brain Res. 266, 83—95.PERKINS, M. N. & STONE, T. W. (1982). An iontophoretic investigation of the actions of

convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid.Brain Res. 247, 184-187.

RANSOM, B. R., BULLOCK, P. N. & NELSON, P. G. (1977). Mouse spinal cord in cell culture. I II .Neuronal chemosensitivity and its relationship to synaptic activity. J. Neurophysiol. 40,1163-1177.

REDMAN, S. (1979). .Functional mechanisms at group la synapses. Prog. Neurobiol. 12, 33-83.RENSHAW, B. (1941). Influence of discharge of motoneurons upon excitation of neighboring

motoneurons. J. Neurophysiol. 4, 167-183.RENSHAW, B. (1946). Central effects of centripetal impulses in axons of spinal ventral roots.

J. Neurophysiol. 9, 191-204.ROBERTS, P. J. (1974). The release of amino acids with proposed transmitter function from the

cuneate and gracile nuclei of the rat, in vivo. Brain Res. 67, 419-428.ROBERTS, P. J., KEEN, P. & MITCHELL, J. F. (1973). The distribution and axonal transport of free

amino acids and related compounds in the dorsal sensory neuron of the rat, as determined by thedansyl reaction. J. Neurochem. 21, 199-209.

SALT, T. E. & HILL, R. G. (1983a). Pharmacological differentiation between responses of ratmedullary dorsal horn neurons to noxious mechanical and noxious thermal cutaneous stimuli.Brain Res. 263, 167-171.

SALT, T. E. & HILL, R. G. (19836). Neurotransmitter candidates of somatosensory primaryafferents fibers. Neurosci. 10, 1083-1103.


SCHNEIDER, S. P. & PERL, E. R. (1985). Kynurenic acid antagonizes synaptic and amino acidexcitation of hamster spinal dorsal horn neurons in vitro. Soc. Neurosci. Abstr. 11, 66.

SHAPOLVALOV, A. I., SHIRIAEV, B. I. & TAMAROVA, Z. A. (1979). Synaptic activity in motoneuronsof the immature cat spinal cord in vitro. Effects of manganese and tetrodotoxin. Brain Res. 160,524-528.

SLADECZEK, F., PIN, J. P., RECASENS, M., BOCKAERT, J. & WEISS, S. (1985). Glutamate

stimulates inositol phosphate formation in striatal neurons. Nature, Land. 317, 717-719.TAKAHASHI, T. (1984). Inhibitory miniature synaptic potentials in rat motoneurons. Proc. R. Soc.

Ser. B 221, 103-109.TAKEUCHI, A., ONODERA, R. & KAWAGOE, R. (1983). The effects of dorsal root stimulation on the

release of endogenous glutamate from the frog spinal cord. Proc. Japan Acad. 59, 88-92.THOMSON, A. M. (1986). A magnesium-sensitive post-synaptic potential in rat cerebral cortex

resembles neuronal responses to N-methylaspartate. J. Physiol., Land. 370, 531-549.THOMSON, A. M., WEST, D. C. & LODGE, D. (1985). AniV-methyl aspartate receptor-mediated

synapse in rat cerebral cortex: a site of action of ketamine? Nature, Land. 313, 479-481.URBAN, L. & RANDIC, M. (1984). Slow excitatory transmission in rat dorsal horn. Possible

mediation by peptides. Brain Res. 290, 336—341.WALMSLEY, B. &TRACEY, D. J. (1981). An intracellular study of the Renshaw cell. Brain Res. 223,

170-175.WATKINS, J. C. & EVANS, R. H. (1981). Excitatory amino acid transmitters. A. Rev. Pharmac.

Toxicol. 21, 165-204.WOMACK, M. D., HANLEY, M. R. & JESSELL, T. M. (1985). Functional substance P receptors on a

rat pancreatic acinar cell line. J. Neurosci. 5, 3370-3378.

Documents

amino acid receptor-mediated transmission at primary afferent