Modulation of excitatory amino acid responses in rat dorsal horn neurons by tachykinins.

JOURNALOFNEUROPHYSIOLOGY Vol. 68, No. 1, July 1992. Printed

in U.S.A.

Modulation of Excitatory Amino Acid Responses in Rat Dorsal Horn Neurons by Tachykinins K. I. RUSIN, P. D. RYU, AND M. RANDIC Department of Veterinary Physiology and Pharmacology,

SUMMARY

AND

Iowa State University, Ames, Iowa 50011

CONCLUSIONS

1. In freshly isolatedspinaldorsalhorn (DH) neurons(laminae

I-III) of the young rat, the effects of tachykinins (substanceP, neurokinin A) on inward current induced by excitatory amino acidswere studiedunder whole-cellvoltage-clampconditions. 2. When the cellswere clampedto a holding potential of -60 mv, a simultaneous application of N-methyl-D-aspartate (NMDA) ( 10e4M) and substanceP (SP) (2 X 10-9-10-7 M) for 10s reversibly enhanced(by 129.6t 8.2%,mean t SE) the peak amplitude of the initial transient component of the NMDA-induced current in -60% of the examinedcellsand reducedit (to 83.3 t 2.7%) in 27% of the cells. In addition, SP produced an increase(by 133.6& 11.7%)or a smalldecrease(to 85.9 ? 1.4%) in the steady-statecomponent of the NMDA response.In differenceto SP, a simultaneousapplication of NMDA ( 10s4M) and neurokinin A (NKA) ( 10-10-10-7M) reversibly suppressed (to 86.8 t 2.1%) the peak amplitude of the NMDA-induced current in 75%of the examinedcells. 3. The NMDA-induced currentswere modulatedby tachykinins not only during the coadministrationbut up to 20 min after the removal of the peptide.SPpotentiated the initial peakNMDA current by 147.9& 8.1YO in 78%of examinedcellsand decreased it (76.3 k 57%) in 11%of cells.The potentiating effect wasconcentration-dependent (range: 10-‘1-10-8 M) and reversible, but it wasreducedwith repeatedapplications.In addition, SPincreased (by 125.4 t 3.6%) or reduced(to 86.0 t 1.8%) the steady-state componentof the NMDA response. 4. When the singleDH neuronswere exposedto SPor NKA for 30 s-7 min before the testingof the NMDA responses, tachykinins had two distinct effectson the peak amplitude of the transientcomponentof the NMDA-induced current, consistingof an initial depression(SP: to 64.8 t 2.1%; NKA: to 76.3 t 4.4%) followed by a potentiation (SP: by 146.6 t 6.8%; NKA: by 178.4t_ 35.2%). The enhancingeffect in somecellslasted5 1 h. 5. A claimed novel nonselectivetachykinin antagonist,spantide II ( 10e8M) coadministeredwith NMDA ( 10B4M), slightly depressed the peak componentof NMDA-induced current. In addition, it effectively blocked the SP-inducedpotentiation of the responses of DH neuronsto NMDA. 6. The developmentof the SPenhancementof the NMDA-induced current is prevented in the presenceof glycine. 7-Chlorokynurenic acid, a competitive antagonistat the glycine allosteric site of the NMDA receptor, led to a reestablishmentof the SP effect. 7. When bis-( o-aminophenoxy)-N, N,N’,N’-tetraacetic acid (BAPTA) concentration in the pipette solution was increased from 1 to 10 mM, both the initial depressanteffect and the late potentiation of the NMDA-induced current causedby SP was noticeably reduced.This result is consistentwith a possibilitythat the SP enhancementof NMDA responsesof DH neuronscould resultfrom a riseof [Ca2+liand subsequentactivation of Ca2+-dependent processes.

8. 4-@-Phorbol12,13-dibutyrate,an activator of protein kinase C, and forskolin, an activator of adenylatecyclase,appliedfor l-4 min, mimic the effectsof tachykinins by producing an initial depressionfollowed by a marked and prolongedpotentiation of the NMDA-induced current responses of isolatedDH neurons.Staurosporine,the agent known to inhibit both protein kinaseC and cyclic AMP-dependent protein kinase, reducedthe tachykinincausedpotentiation of the NMDA response.Theseresultssuggest that in the rat spinal DH protein kinaseC and the cyclic AMP systemmay both be involved in the regulation of postsynaptic NMDA receptor function. In addition, phosphorylation of the NMDA receptor-ion channel complex or a related protein by a staurosporine-sensitive protein kinase(s) may be involved in the mediation of the tachykinin effects. 9. SP(2 X lo-“-2 X low6M) applied for 30 s-2.5 min produceda smallincreasein the transient and sustainedcomponents of quisqualicacid-induced currents in a proportion of DH neurons. Pretreatmentwith SPor NKA (5 X 10-10-10-8M) for l-5 min enhanced(SP: by 118.3* 4.0%;NKA: by 134.0t 12.2%)the amplitude of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( AMPA)-induced current in 42% of the testedcells. However, KA-induced currents appearto be little affectedby tachykinins. 10. Our resultssuggestthat tachykinins modulatethe NMDA and the AMPA receptors,signalingfunction in a proportion of the rat spinal DH neurons.Possiblemechanismsof action are discussedin relation to tachykinin-dependent regulation of excitatory amino acid-mediated primary afferent neurotransmission, including nociception.

INTRODUCTION

The spinal dorsal horn (DH) is an area where primary afferent fibers arising predominantly from skin, but also the viscera and muscles, terminate and form the first synaptic relay with dendrites of DH neurons. For this reason, the DH has been regarded as an important site for the initial processing of signals directly related to the transmission and modulation of cutaneous information, including pain. Experimental evidence indicates that there are two major classes of chemical compounds that are released during activation of primary afferent fibers in the mammalian DH. Dicarboxylic amino acids, glutamate, and aspartate are the major candidates for the fast excitatory neurotransmitters (Gerber and Randic 1989; Kangrga et al. 1988; Mayer and Westbrook 1987b; Yoshimura and Jesse11 1990), whereas tachykinins (SP, neurokinin A) appear to be functionally involved in the slow excitatory synaptic transmission (Urban and Rand2 1984).

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266

K. I. RUSIN, P. D. RYU, AND M. RANDIC

Neuronal excitatory amino acids ( EAAs), including glutamate and aspartate, produce their effects through two broad categories of receptors called ionotropic and metabotropic (Schoepp et al. 199 1; Watkins et al. 199 1) . The ionotropic-N-methyl-D-aspartate (NMDA) , a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) / quisqualate ( QA) , and kainate (KA)-receptors directly regulate the opening of ion channels to Na, K+, and, in the case of NMDA receptors, to Ca2+ as well (Ascher and Nowak 1988b; Mayer and Westbrook 1987a). In addition, the activation of ionotropic receptors can induce Ca2+ influx through voltage-dependent Ca2+ channels activated as a result of cell depolarization. Their activation is responsible for synaptic currents underlying fast excitatory synaptic transmission. The metabotropic receptors (activated by glutamate/ QA) appear to be coupled to phospholipase C through G proteins. Their activation causes an increase in turnover of polyphosphoinositides (Nicoletti et al. 1986a,b; Sladeczeck et al. 1985 ) and release of Ca2+ from intracellular stores. Thus the activation of both classes of EAA receptors can result in elevation of intracellular free Ca2+ concentration ([ Ca2+li) (Mayer and Miller 199 1). The increase in [ Ca2+li, in turn, may lead to activation of other second messenger systems. Such changes in second messengers may lead to changes in the properties of EAA receptor channel complexes that contribute to slow excitatory synaptic transmission (Charpak and Gahwiler 199 1) and to long-term influences on the fast excitatory synaptic transmission, neuronal excitability (Malenka et al. 1988; Mayer and Miller 199 1) , nerve cell architecture ( Lipton and Kater 1989), and gene expression (Szekely et al. 1989). Substance P (SP), the most extensively characterized member of tachykinin peptide family, has been demonstrated to depolarize DH neurons, to increase [Ca2+li in these neurons ( Womack et al. 1988, 1989) and to stimulate phosphoinositide turnover (Mantyh et al. 1984). A stimulating effect of SP on the low- and high-voltage activated Ca2+ channels has been reported (Ryu and Randic 1990), and SP is shown to modify a number of voltage-dependent potassium currents (Murase et al. 1989a). Immunocytochemical studies have demonstrated that glutamate coexists with SP in many small dorsal root ganglia neurons and their terminals in the superficial laminae of the rat spinal DH ( DeBiasi and Rustioni 1988). However, the physiological significance of this phenomenon for primary afferent neurotransmission is not understood. We have recently reported that SP increases glutamate-induced currents in acutely isolated rat spinal DH neurons (Randic et al. 1990). However, the specific subtype(s) of glutamate and tachykinin receptors involved and the cellular mechanisms underlying the enhancement of glutamate receptoractivated conductance have not been as yet elucidated. The primary objective of the present study was to determine the pharmacological specificity of the SP effects on EAA responses of rat spinal DH neurons (L,-L,) by the use of selective agonists of EAA receptors such as NMDA, AMPA/QA and KA. The use of freshly isolated DH neuron preparation permitted observation of the direct interaction between tachykinins and EAA agents on an identifiable preparation of central neurons by eliminating possible confounding synaptic interactions (Murase et al. 1989b).

The results show that the major effect of tachykinins (SP, neurokinin A) is the potentiation of NMDA- and to a smaller degree QA- and AMPA-induced inward current responses of a proportion of DH neurons. The results are consistent with a possibility that tachykinins might modulate the NMDA receptor-ion channel complex by interacting with Ca2+ -dependent cellular processes that regulate the function of NMDA receptor channels. Part of these results has been presented elsewhere (Hecimovic et al. 1990; Rusin et al. 1991b). MATERIALS

AND

METHODS

Cell isolation Single spinal DH neuronsin the Rexed’s laminae I-III were isolatedacutely from 9- to 16-day-oldSprague-Dawleyratsby the methoddescribedelsewhere (Murase et al. 1989b,1990;Randicet al. 1990). Briefly, transverseslices( 350 pm) of the lumbar spinal cord were cut with the useof a Vibratome (Murase and Randic 1984)and a modified artificial cerebrospinalfluid in which NaCl wasreplacedby sucrose( Aghajanian and Rasmussen 1989). The superficialdorsal horn (SDH) region in the sliceswasthen dissectedout and incubatedin a bicarbonate-bufferedsolution containing (in mM) 124NaCI, 5 KCl, 1.2 IQ&PO,, 1.3MgSO,, 2.4 CaCl,, 26 NaHCO,, and 10 glucose,oxygenatedby 95% 02-5% CO2at 35“C containing pronase(0.1 mg/ ml, Calbiochem)for 10 min, thermolysin or proteasetype X (0.06 mg/ml, Sigma) for another 10 min, and ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA, 1.OmM) and nominally zero Ca2+for 4 min. After the treatment with the enzymes,the SDH wasmechanicallydissociatedin a culture dish(Falcon 3801) filled with a N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-buffered solution containing (in mM) 150 NaCl, 5 KCl, 2 CaCl,, 1MgCl,, 10HEPES, 10D-glucose,NaOH to adjust pH to 7.4, and bovine serumalbumin 0.1 mg/ml, saturatedby 100%OZ. The disheswere placed in an O,-filled incubator and kept at room temperature (20-23°C) until recording. Isolated cellsadheredto the bottom of the culture dishwithin 1 h, andthen the recording started.They remain in a good condition for 18 h, exhibiting a variety of voltage-dependentionic currentsand having good responses to EAAs and peptides(Murase et al. 1989b).

Recording conditions For recording, a dish wasset on a stageof an inverted microscopeand continuously superfused( l-2 ml/min) with a nominally O-Mg2+solution containing (in mM): 150NaCl, 5 KCl, 0.5 or 2 CaCl,, 10 HEPES, 10 D-glucose,NaOH to adjustpH to 7.4, and alsotetrodotoxin ( 5 X 1O-’ M). Osmolality of the solutionto 320-335 mosmolwasadjustedwith sucrose.In a majority of experiments,nominally glycine-free solutions,which are likely to contain 120 nM glycine (the deionized water used to prepare recording and testing solutions contains a background glycine concentration of -20 nM, as determined by high performance liquid chromatography), were used.However, in someof the experiments,the extracellular solution contained 5 X 10W8-10m6M glycine (Sigma) to producea potentiation of the NMDA response (Johnsonand Ascher 1987). The whole-cell voltage-clamptechnique (Hamill et al. 1981) wasusedto record membranecurrents of isolatedDH neuronsat room temperature( 20-23 “C) . Macroscopiccurrents, elicited by EAA and tachykinins, and holding voltagesweremonitored continuously with a List L/M-EPC7 patch-clampamplifier (Medical Systems,Greenvale, NY). Current signalswere filtered with a low-passfilter (300 Hz), and current and voltage signalswere

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MODULATION

OF EAA

RESPONSES BY TACHYKININS

recordedon a Gould-Brusch (model 2400s) pen recorder. In addition, in 31 cells(20 experiments), currents werefiltered with an external eight-poleBessel1 filter setat corner frequency 2 kHz (-3 dB); current signalswere digitized at 2 ms/point and storedon a Nicolet-4094 digital oscilloscopediskettefor later analysis.In the majority of experiments,current measurements were madefrom the chart paper.The electrodeswereconstructedfrom borosilicate thin-walled glass(Boralex, RochesterScientific, Rochester,NY). A modifiedDavid Kopf Instruments700Cvertical puller wasused to pull electrodesof an approximately uniform tip diameter( 1S2.0 pm) and shape.Each electrodewasthen fire-polished,and a concertedeffort wasmadeto keepthe electrodetip diameterand geometryasconsistentaspossible.Electrodeswerefilled with one of three internal solutions containing (in mM) 120 potassium aspartateor gluconate,20 KCl, 10NaCl, 1MgC&, 0 or 0.5 CaCl,, 10HEPES, 1 or 5 EGTA, 3 NaATP or MgATP, 0.1-0.3 GTP, 0.1 leupeptin and tris( hydroxymethyl)aminomethane (TRIS) base for pH 7.2. In someexperiments,we replacedpotassiumaspartate ( 120 mM) and KC1 (20 mM) with CsCl ( 140 mM). Internal solutionscontaining different anionsgavesimilarresults.The pHinsensitive calcium buffer 1,2-bis-(2-aminophenoxy ethane) N,N,N’,N’-tetraacetic acid, (BAPTA, Sigma) (Tsien 1980) was substitutedin someexperimentsfor EGTA. Once filled with an intracellular solution, electrodeshad resistancesof 5- 10 Mtlt. Membrane potential wasclampedat between-70 and -50 mV, where no holding current (leak cu~ent) was usually observed. Recordingswere made from phase-brightcells free of apparent membraneblisters.Whole-cellrecordingswereundertakenonly if membranepotential was not lessnegative than -40 mV and if action potential amplitude exceeded80 mV. Cellsincludedin this study were recordedfor extendedperiodsrangingfrom 30 min to 3 h and did not demonstrateswellingor other grossmo~hological changesduring the recording period. In addition, little or no leak or noise current was detected in thesecellseven after l-2 h of recording. Cells that failed to meet these criteria were not included.

Appli~utio~s

of excitatory umi~o acids aid peptides

267

taken to prevent contamination of solutionswith glycine by the useof disposableglasswareand covering solutions.EAA receptor agonistsand antagonists[6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5.phosphonovalerate(D-APV)] were obtained from Sigma, Cambridge Research Biochemicals, and Tokris, SPand neurokinin A werefrom CambridgeResearchBiochemicals,Sigma, and Peninsula,whereasspantide II, (D-NicLys ‘, 3-Pa13,D-Cl,Phe5, Asn6, D-Trp7$9,Nle i ’ )-SP, wasa gift from Dr. K. Folkers(University of Texasat Austin, USA). Stock solutionsof phorbol esters[4/3-phorbol-12,13=dibutyrate( PDBu ) and4a-phorbol-12,13-didecanoate (4ac-PDiDec)]of 1Ov3M were madein dimethyl sulfoxide and then frozen in aliquotsto be used in singleexperiments.The aliquotswerediluted in perfusingsolution beforeadministration.Chemicalsusedand their sourceswere asfollows: phorbol esters( Sigma), forskolin (Calbiochem), staurosporine(Calbiochem).

Dutu dialysis To compareresponses betweendifferent cells,the peak amplitude of inward current at any given time was normalized with respectto amplitude of the control response.Population results are expressedasmeanst SE. RESULTS

General observutio~s Whole-cell voltage-clamp recordings were made from a total of -250 acutely isolated rat spinal DH neurons, mostly from laminae I-III. Examination of cells under phase-contrast optics revealed variation of soma shape and size. However, most cells appeared roughly spherical or oval, measuring on average - I 1 X 16 pm diam. Isolated DH neurons preserved between 36.0 and 70.0 pm of the proximal dendrites. Under a whole-cell current-clamp condition, the isolated cells had resting membrane potentials in the range of -40 to -60 mV. The cells generated shop-lasting action potentials, 80-A 00 mV in amplitude, followed by fast and slow hyperpolarizing and depolarizing afterpotentials (Murase et al. 1989b). These values are similar to those obtained in the spinal DH slice preparation with the use of intracellular microelectrodes (Murase and Randic 1983, 1984). Measurement of the zero current potential under whole-cell voltage-clamp with standard solutions gave similar values in the range of -40 to -60 mV. However, such values may not correspond to the true cell resting membrane potential for several reasons. First, it is generally accepted that intracellular recording with microelectrodes in small cells is frequently associated with cell membrane damage. Second, inte~retation of patch-clamp measurement of resting membrane potential has two shortcomings: I ) spontaneous fluctuations are frequently observed in cells with high cell membrane resistance (Fenwick et al. 1982)) and 2) the normal intracellular milieu may be significantly altered as a result of cell dialysis. No detectable spontaneous activity in the form of action potentials was observed under whole-cell recording mode.

Excitatory amino acids (sodium salts) and tachykinins (SP, neurokinin A) weredissolvedin the HEPES-bufferedsolution and applied by a pressuresystem,the major part of which was a Yshapedtube (Murase et al. 1989b,1990). The orifice of the Y-tube tip waspositioned300-500 pm away from a cell that wasalways exposedto a rapid flow of the external solution or the drug-containing solution,both of which wereappliedfrom the sameorifice. Exchangespeedof the external solution surroundinga singlecell wasmeasuredby the risetime of current responses inducedby the application of KA- or glutamate-containingsolution or by a decline of KA-induced currentson replacementof Na+ in perfusing solution by TRIS. It rangedbetween50 and 100ms, mostly depending on the distance betweenthe cell and the orifice of the Y-tube tip and on the level of solutions.The flow rate of solution running out from the Y-tube tip was - 1.5 ml/min. Using this technique, we could routinely evoke reproducible NMDA currentsthat peakedat - 100ms. EAA and peptideswereapplied at low frequencies(0.007 Hz) to minimize desensitization.Only one cell in a dishwastestedwith a peptide;the number of peptide administrationsdependedlargely on the developmentof desensitization. All the HEPES-bufferedsolutionswere madeup with a purified water (resistance- 18MQ/cm) that wasdouble-distilledand then membrane filtered (Millipore). To avoid degradation of labile components in the solutions [peptides, ATP, guanosineS-tri- Inwurd currents induced by excitatory amino acids phosphate(GTP), leupeptineJ they were either made freshly beGlutamate is considered to be a mixed agonist in that it fore the beginning of the experiment or were aliquoted into smaller volumes and frozen until they were used. Caution was activates multiple receptors defined by the actions of selec-



268

tive agonists such as NMDA, AMPA, and KA (Watkins et al. 199 1). In confirmation of a previous report, pressure ejection of NMDA, AMPA, QA, and KA induced inward currents in a majority of freshly isolated DH neurons (Murase et al. 1989b), indicating the presence of three subtypes of EAA receptors (Cotman and Iversen 1987; Mayer and Westbrook 1987b; Watkins et al. 199 1). The currents activated by NMDA at a concentration of 550 PM were nondesensitizing and inward when the membrane potential was held at -60 mV. Higher concentrations of NMDA (2 10S4) elicited a short-latency fast transient inward current that partially desensitized to a steady level (Fig. 1). At a holding potential of -60 mV, initial peak NMDA ( 10S4 M) currents ranged from -20 to -560 pA. The amplitude of the steady-state current component elicited by 1Om4 M NMDA was on average -20% of the initial peak value, although the ratio varied in different cells. As shown in Fig. 1, the gradual and progressive decrease in the peak amplitude of the NMDA responses occurred during the first 20-25 min of recording. Thereafter, the responses were stable at -76% of the initial amplitude when CsCl intracellular dialysis solution was used (Fig. 1A) and at -63%

when potassium aspartate solution was employed (Fig. 1 B). Both initial peak and steady-state components of the NMDA-induced currents showed some typical characteristics of NMDA channels (Kemp et al. 1987; Thomson 1989; Watkins et al. 199 1); they were potentiated by glycine (Johnson and Ascher 1987; Murase et al. 1989b, Figs. 10 and 1 1 ), reduced by Mg2+ in a voltage-dependent manner (Mayer et al. 1984; Nowak et al. 1984; Rusin and Randi 199 1) and blocked by the competitive antagonist 2-amino5-phosphonovalerate ( 10w5- 10m4 M) (Murase et al. 1989b; Rusin and RandZ 199 1; Watkins et al. 199 1) and the noncompetitive antagonist 6-cyano-7-nitroquinoxaline-2,3dione (Honore et al. 1988; Watkins et al. 199 1). Two other EAA receptor agonists, QA ( > 10 -6 M, Fig. 14)) and infrequently (in 3 of 23 cells) AMPA ( 3-50 PM), produced fast and rapidly desensitizing inward currents after close-range pressure application. In contrast, application of KA to the DH neurons resulted in the initiation of inward current that showed almost (and in many cells completely) no desensitization after reaching its maximum value (Fig. 14B). At a holding potential of -60 mV, the peak amplitude of inward currents elicited by 10 PM of QA, AMPA, and KA ranged

A Y

r(

c

-I

25 pA

10s

A o-O-0 B .-0,

‘O-o-0-0 0

‘0,

O-o~-o-0

FIG. 1. Stability of peak N-methyl-D-aspartate (NMDA) currents in 2 different freshly isolated rat DH neurons in the presence of intracellular dialysis solution containing ( in mM ): 3 MgATP, 0.1 GTP, and 0.1 leupeptin. A and B: typical whole-cell records of inward NMDA currents generated by applications of 0.1 mM NMDA for 10 s are shown continuously in a pen recorder trace. These applications were repeated once every 2.5 min and commenced immediately after the patch was ruptured. The holding potential in this and subsequent figures was -60 mV. Note the gradual and progressive decrease in the peak amplitude of the responses during the first 15 (B) and 23 min (A) of recording. Thereafter, the responses were relatively stable at ~76% (A ) and 63% (B) of the initial amplitude. C: results from A and B illustrated graphically. In A, the intracellular solution contained (in mM) 140 CsCl, 10 NaCl, 1 MgCl,, 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and 1 ethylene glycol-bis( ,8-aminoethyl ether)-N,N,N: N’-tetraacetic acid (EGTA). In B, we replaced CsCl ( 140 mM) with potassium aspartate ( 120 mM) and KC1 (20 mM). 1l-day-old rat.


MODULATION

OF EAA RESPONSES BY TACHYKININS

269

from 50 to 1,640 pA for QA (n = 21), lo-375 pA for AMPA (n = 19), and lo-120 pA for KA (n = 23). The receptors associated with QA and KA channels showed pharmacological properties of the non-NMDA receptor subtypes, as indicated by the efficacy of the competitive antagonist ( Honore et al. 1988 ) CNQX ( 1- 10 PM) to block the activity. In contrast, CNQX exerted noncompetitive antagonism at NMDA receptors, acting via the modulatory glycine site of the NMDA receptor (Watkins et al. 199 1).

(n = 37; Fig. 2A) and reduced it (to 83.3 t 2.7%) in 27% of the cells relative to control (control values expressed as 100% ) . In addition, SP produced an increase (by 133.6 t 11.7%; n = 7) or a small decrease (to 85.9 t 1.4%; n = 10) in the steady-state component of the NMDA response. The NMDA response in five remaining cells was unaffected by SP. The minimum effective concentration of SP for the enhancing effect was 10 -I ’ M; however, the peak amplitude of the NMDA-induced current did not usually increase further with increases in SP concentration beyond 10s7 M Effects of a simultaneous application of NMDA and (Fig. 2B). In difference, no concentration dependence was tachykinins observed for the depressant effect of SP. There was no apparWe have recently reported that glutamate-activated con- ent correlation between the incidence of cells showing the ductance in freshly isolated rat spinal DH neurons is en- SP potentiation or depression of the NMDA responses and hanced by SP ( Randic et al. 1990), but the specific subtypes the cell types isolated. of glutamate and tachykinin receptors involved and the celThe NMDA-induced currents were modified by SP not lular mechanisms underlying the SP enhancement have not only during the coadministration but 520 min (9.0 t 1.9, been as yet elucidated. In this section, we present results n = 13) after removal of the peptide (Figs. 2 and 3, Table carried out to analyze direct postsynaptic interactions be- 1). SP potentiated the initial peak NMDA current (by tween NMDA and tachykinin receptors in DH neurons. To 147.9 t 8.1%, n = 29) in 78% of the examined cells and investigate the effects of tachykinins (SP, NKA) on the decreased it (to 76.3 t 5.7%) in four cells, whereas four cells NMDA-induced currents, we used two different protocols: were not affected. As shown in Fig. 3C, the potentiating 1) a simultaneous application of 0.1 mM NMDA and pep- effect of SP was concentration dependent (range: 10-l ‘tide ( 10-l’ - 10 -6 M) for 10 s; and 2) perfusion of a cell with 10 -* M ) and reversible, but it desensitized with repeated a peptide (5 X 10-l’ M-10s7 M) for 30 s-7 min before applications (Fig. 2, C and D). In this instance, the term starting the applications of NMDA at 2.5-min intervals for desensitization refers mainly to the empirical observation 260 min. Because the progressive decrease in the peak am- of a decreased responsiveness rather than the specific mechplitude of the NMDA response occurred during the first anism by which the sensitivity to agonist application is re15-20 min of recording (Fig. 1)) the peptide application duced. Furthermore, SP increased (by 125.4 t 3.6%, n = 9) did not begin for 220-25 min after the patch was ruptured or reduced (to 86.0 t 1.8%, n = 6) the steady-state compoand only when the cell showed three stable responses to nent of the NMDA response in 4 1% of the tested cells. BeNMDA. In a majority of the experiments, a nominally gly- cause this effect was observed in a relatively small number tine-free perfusing solution containing 5 X 10 -7 M tetrodoof cells, further analysis was not performed. toxin (TTX) was used. There is evidence for the presence of at least three differWhen the microelectrode solution contained 1 mM ent types of receptors for tachykinins both in CNS and peEGTA or BAPTA and the bath solution contained either ripheral tissues (Lee et al. 1986; O’Donohue et al. 1987; 0.5 or 2 mM Ca2+, SP had two effects on the NMDA-inRegoli et al. 1987). The neurokinin receptors NK- 1, NK-2, duced current, as shown in Table 1. When the cells were and NK-3 are preferentially activated by the endogenous clamped to a holding potential of -60 mV, a simultaneous peptides SP, neurokinin A (NKA) and neurokinin B, reapplication of NMDA ( low4 M) and SP (2 X 1Ov9 - 1OB7 spectively (Lee et al. 1986). To determine the subclass of M) for 10 s reversibly enhanced the peak amplitude of the tachykinin receptors involved in the SP potentiation of the initial transient component of the NMDA-induced current NMDA response, we compared the effects of SP, which acts (by 129.6 t 8.2%, mean t SE) in 60% of the examined cells primarily at the NK-1 class of tachykinin receptors, with TABLE

1.

Modulation of NMDA-induced current during and after a simultaneousapplication of NMDA and tachykinins

Types of Modulation Peak Initial effect Late effect Steady-state Initial effect Late effect

Neurokinin

Substance P (n = 37)

A (n = 12)

Increase

Decrease

No change

Increase

Decrease

No change

60 (129.6 AI 8.2) 78 (147.9 + 8.1)

27 (83.3 + 2.7) 11 (76.3 + 5.7)

13

17 (137.0 AI 29.0) 83 (124.9 f: 5.5)

75 (86.8 It 2.1) 17 (77.5 2 4.5)

8

19 (133.6 t 11.7) 24 (125.4 t 3.6)

27 (85.9 t 1.4) 16 (86.0 t 1.8)

54

42 (129.0 + 8.9) 33 (137.8 t 7.2)

50 (80.0 ziz 3.7) 33 (83.3 t 5.5)

8

11

59

0

33

Values are percent of cells showing a particular type of interaction between NMDA and tachykinins; values in parentheses are percent change (means * SE) in the initial transient component (peak) and the steady-state component (steady-state) of NMDA-induced current in the presence (initial effect) and after removal (late effect) of substance P or neurokinin A. NMDA, N-methyl-o-aspartate.


270


A [NMDA]

C

SP 2 nM 0-

m(

3 min -

SP 2 nM

6 min -

0

-

-

3 min -

8 min -

f

B

SP -

, 25 pA

25 pA

10s

a

10s

b

FIG. 2. N-methyl-D-aspartate (NMDA) responses in DH neurons are potentiated by substance P (SP), and the effect shows dose dependence and desensitization. Effects of SP on membrane currents induced by NMDA in 3 different acutely isolated rat spinal DH neurons are shown. Two different DH neurons were voltage-clamped to -60 mV, and initial transient and steady-state components of inward current measured at the peak and at the end of current excursion generated by 10-s application ofO.1 mM NMDA alone or 0.1 mM NMDA plus 2 nM SP in A, or 0.1 mM NMDA plus 1, 10, or 100 nM SP in B, as indicated. Bars: period of a rapid application of chemicals. Initial peak NMDA current is increased when SP is present and also after the removal of the peptide (A). NMDA responses in another DH neuron (b) are potentiated by SP (Cand D), and the effect showed desensitization (D). Individual NMDA responses after SP application, at the times indicated, are shown. The perfusion solutions contained 1 mM Ca*+ in A, 2 mM Ca’+ in B, and 0.5 mM Ca2+ in Cand D, and no added Mg*+ or glycine. Pipette solution contained I mM bis-(o-aminophenoxy)-N,N,N’,N’-tetraacetic acid (BAPTA, A, C, and D) or 1 mM ethylene glycol-bis(&aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA, B). Tetrodotoxin (TTX, 5 X lo-’ M) was present throughout. A: 1 l-day-old rat; B: lo-day-old rat; C and D: lCday-old rat.

that of NKA, which has a higher affinity than SP for NK-2 and NK-3 receptors. In difference to SP, a simultaneous applicationofNMDA( 10d4M)andNKA( 10-‘“-10-7M) reversibly suppressed (to 86.8 + 2.1%) the peak amplitude of the NMDA-induced current in 9 of 12 examined cells. In two cells, however, NKA augmented (by 137.0 + 29.0%) the peak NMDA response in comparison to control, whereas one cell was not affected. In addition, both increases (by 129.0 + 8.9%, n = 5) and decreases (to 80.0 k 3.7%, y1= 6) of the steady-state component were observed (Table 1). Similar to SP (Fig. 3B), the NMDA-induced currents were modified by NKA not only during coadministration but 118 min (8.8 + 1.1, y1= 10) after removal ofthe peptide (Fig. 3A, Table 1). NKA potentiated (by 124.9 ZL 5.5%) the initial peak NMDA response in 10 of 12 tested cells and reduced it (to 77.5 t 4.5%) in 2 cells. In addition, NKA produced an increase (by 137.8 ?Z 7.2%, n = 4) or a decrease (to 83.3 +- 5.5%, y1= 4) in the steady-state component of the NMDA response.

Because pronase and thermolysin were used in the dissociation procedure to exclude the possibility of an alteration of cell responses to NMDA and tachykinins by proteolysis, the actions of tachykinins were tested on NMDA-induced current in exclusively mechanically isolated cells. As shown in Fig. 4, the potentiating effect of NKA was also seen in a mechanically isolated DH neuron. The results obtained with SP and NKA suggest that the effects of the tachykinins on the NMDA receptors are mediated through the activation of both NK-1 and NK-2 receptors.

Eflects of tachykinin receptor antagonist spantide II We next examined the action of a claimed novel nonselective tachykinin antagonist, spantide II (Folkers et al. 1990)) on the SP-induced potentiation of NMDA-induced currents. Application of spantide II ( 10m8 M), which alone had only a small depressant effect (to 86.3 + 2.9%, n = 6) on the NMDA response (Fig. 5A, top), prevented the SPcaused enhancement of the NMDA-activated current both


MODULATION


C

A NKA 0.1 nM [NMDA]

271

160-

8 min

13 min

E ‘z k! = 140--

-

0-0 o-o

NKP sp

I

0

i3

-12

-11

-10

-9 Log

-8 [Peptldel

04

D 2 nM

3 min

I

I

8 min

10s

3. Substance P (SP) and neurokinin A (NKA) increase N-methyl+aspartate (NMDA) responses of DH neurons, and the effects are concentration dependent. Traces show inward current responses induced by 0.1 mM NMDA in a DH neuron (D) before, during, and after combined administration with NKA (0.1 nM in A) or SP (2 nM in B). Holding potential -60 mV. Peak inward current induced by NMDA was slightly reduced during coadministration of NKA + NMDA, but potentiated thereafter (A). In the same neuron, the peak ofNMDA-induced inward current was increased by SP both during and after the removal of the peptide (B). The perfusing solution contained 0.5 mM Ca’+ and pipette solution 5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). Tetrodotoxin (TTX, 5 X IO-’ M) was present throughout. 1 l-day-old rat. B: concentration dependence of the SP- or NKA-induced enhancement of NMDA-induced currents. Peak inward currents induced by 0.1 mM NMDA in the presence of various concentrations of SP or NKA were plotted. Results are expressed as the mean percentage of the control current responses evoked by NMDA before the addition of tachykinins. Each point is an average of data obtained from 3-20 cells, and the vertical bars represent SE. FIG.

during (initial effect) and after removal (late effect) of the peptide (Fig. 5A, middle). After the removal of Spantide II, the same concentration of SP (2 X 1O-9 M) produced a significant increase in the NMDA response (Fig. 5A, bottom). This antagonistic effect of Spantide II, observed in six DH neurons, is summarized in Fig. 5 B. This finding suggests that the effect of SP on the NMDA-activated conductance is a true tachykinin receptor-mediated event.

Efects ofpretreatment of DH neurons with tachykinins on the NMDA-induced current Because the tachykinins (SP, NK A ) and NMDA were applied simultaneously in the experiments discussed in previous sections, the time course of the potentiating effect could be influenced by a slow onset of action of the peptides in relation to NMDA. Therefore additional experiments were performed in which the single DH neurons were exposed to SP (2 X 10-9-10-7 M) or NKA (2 X 10-9-10-7 M) for 30 s-7 min before the testing of the NMDA responses to preequilibrate the presumable peptide binding. Using this protocol, we found (Fig. 6, Table 2) that the

tachykinins had two distinct effects on the peak amplitude of the transient component of NMDA-induced current consisting of an initial depression (SP to 64.8 + 2.1%, n = 66; NKA to 76.3 a 4.4%, n = 8) followed by a marked potentiation (SP by 146.6 f 6.8%, n = 50; NKA by 178.4 & 35.3%, n = 11). The potentiation of the NMDA-induced current occurred in 54% (n = 93 ) of tested cells with SP and 73% (n = 15 ) of the cells examined with NKA. The time course of the SP-induced potentiation of the NMDA-induced current responses in DH neurons appears to be dependent on both concentration and the duration of peptide application. On average, the enhancing effect of SP lasted 13.2 + 1.4 min (n = 26) and that of NKA 25.7 + 2.7 min (n = 6). In a smaller proportion ofthe examined cells, however, an initial increase (SP: 126.6 + 6.1%, n = 11; NKA: 133.0 + 12.8%, n = 4) followed by a decrease (SP: to 70.4 + 3.1%, n = 2 1) of the initial peak amplitude of NMDA current was observed with tachykinins. The steady-state component of the NMDA-induced current was either increased (SP: 142.4 + 11.4%, n = 7; NKA: 129.4 + 14%, n = 5 ) or decreased ( SP: to 7 1.8 + 4.6%, n = 8) . Twelve cells with SP, and three cells with NKA, were not affected.


272

K. I. RUSIN, P. D. RYU, AND M. RANDIC NKA + A (NMDAJ

2

1 m

O-O l -

l

-

3 -

4 -

peak steady-state

‘O\

4 O----O

I ..-•-•@-O-O-O-O-.-.-O I. II 11

0’ -10

0

10

NKA 10 nM

20

30

I1 40

-I 50 min

4. N-methyl-D-aspartate ( NMDA )-induced current responses are also potentiated in an exclusively mechanically dissociated spinal DH neuron from a lo-day-old rat. Traces show inward current responses induced by 0.1 mM NMDA in a DH neuron before, during, and after application of 10 nM neurokinin A (NKA, A). Holding potential -60 mV. Time courses of the peak and steady-state NMDA current responses are shown in the graph (B). The perfusing solution contained 2 mM Ca2+, but it was devoid of bovine serum albumin (BSA) and tetrodotoxin (TTX); the pipette solution contained 1 mM ethylene glycol-bis ( @-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA). FIG.

Efects of tachykinins on NMDA responseof Cs+-loaded cells We have reported that SP and NKA increase the amplitude of both the low-threshold and the high-threshold Ca2+ current in immature rat DH neurons when in vitro spinal cord slice preparation was used (Murase et al. 1989a; Ryu and Randic 1990). In addition, in freshly isolated myocytes of rabbits, SP is known to increase the open probability of Ca2+-activated K+ channels via the activation of L-type Ca2+ channels (Mayer et al. 1990). Therefore, to isolate the tachykinin effects from the effects on the Ca2+-activated K+ conductance, we used intracellular dialysis solution containing Cs+ instead of K+ ions. Cesium has been shown to be impermeable through Ca2+-activated K+ channels. When potassium aspartate and KC1 in the microelectrode solution were replaced by CsCl, a simultaneous application of NMDA and tachykinins (Fig. 7, A and B) and also preincubation (Fig. 7, Cand D) of DH neurons either with SP or NKA produced a marked potentiation of the transient component of NMDA-induced current.

SP-induced inward current When the microelectrode solution contained high K+ with 1 mM EGTA or BAPTA (no added Ca2’) and the bath solution was 150 mM NaCl Ringer solution (containing 2 mM Ca2+ and 5 x lOA7 M TTX), SP (2 X lo-‘O-2 X

10T6 M for 30 s-5 min) by itself induced in 58% of the examined cells (n = 69)) at holding potential of -60 to -70 mV, a transient inward shift in the holding current ( -3.8 t 0.8 pA; range: 1-12 PA) that reached a peak within 2-l 5 s and decreased in the continued presence of the peptide (Fig. 8A). The transient nature of the inward current recorded in response to maintained peptide application may reflect the desensitization of SP receptors, the inactivation of other cellular components involved in the transduction of SP actions, or the depletion of intracellular Ca2+ stores. In addition, only in 1 of 69 cells, an outward shift in the holding current was recorded in response to the application of SP. The SP-induced inward current was accompanied either by an increase in membrane current noise in 67% of the examined cells (Fig. 8 B) or a decrease in 12% of the cells (Fig. 8C). In the whole-cell recording mode, at the resting membrane potential, inward current steps (events) of 1.53.8 pA were most commonly observed. However, in a smaller proportion of cells, the channels corresponding to the larger inward current steps ( 15-50 pA, n = 13) were observed, and SP (2 X 10 -9-2 X 10 -7 M ) markedly altered their frequency. Fig. 9A shows the inward current steps before and after exposure to 2 nM SP. Before the SP application, there were at least five inward current steps recorded (Fig. 9 B). Pressure application of SP resulted in a pronounced increase in the number of inward current events, and characteristically they showed bursting and / or clustering behavior. The maximal increase in the frequency of inward current steps coincided with the peak increase of the NMDA-induced current at 3 min after exposure to SP (Fig. 9B). When Cs+ was substituted for K+ in the pipette solution, SP at holding potential of -60 mV produced an inward shift in the holding current and activated large inward current ( 15-50 PA) steps. This inward current could be a current through Ca2+ channels; however, its voltage- and time dependence and its pharmacology were not studied in the present work. Low- and high-voltage-activated calcium currents have been described in rat DH neurons (Huang 1989; Ryu and Randic 1990), and both were shown to be enhanced by SP (Ryu and Randic 1990).

Interaction of glycine with the SP-induced potentiation of NMDA-induced currents Glycine is known to enhance the activity of NMDA channels, and the strychnine-resistant, high-affinity glycine binding site is located at the NMDA receptor--ion channel complex (Johnson and Ascher 1987; Kushner et al. 1988). Glycine is thought to act either as a coagonist (KIeckner and Dingledine 1988) and/or a regulator of the rate of desensitization at NMDA receptors (Mayer et al. 1989; Vyklick9 et al. 1990). In freshly isolated rat spinal DH neurons, glycine enhances both the initial transient and steady-state components of NMDA-induced current (Murase et al. 1989b; Rusin and Randic 199 1) . To determine whether the interaction of tachykinin receptors with the glycine regulation of NMDA receptor-ion channel complex may be one of possible molecular mechanisms underlying the SP enhancement of the NMDA receptor-activated conductance,


MODULATION


213

A

[NMDA]

s II 1OnM m -

SP 2 nM+S

3 min

5 min

-

0

SII

Is3 m

s II + SP SP

II

5

8 min -

-

100

ki! 5 0

INITIAL

EFFECT

IATE

EFFECT

FIG. 5. Spantide II (SII) prevents the development of the SP enhancement of iv-methyl-D-aspartate (NMDA)-induced currents. A: inward current responses induced by 0.1 mM NMDA recorded at 2-min intervals before, during, and after the combined administration with spantide II (SII, top traces) or substance P (SP) plus SII (middle traces) or SP (bottom traces) from the same cell (Inset) held at -60 mV. SII ( 10 nM) alone, a novel nonselective tachykinin antagonist, slightly suppressed the peak NMDA-induced current. In the presence of SII, the SP (2 nM) potentiation of NMDA-induced current was not observed. The perfusing solution contained 0.5 mM Ca*+, and the pipette solution contained 5 mM ethylene glycol-bis( @aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). 13-day-old rat. B: absence ofthe SP potentiation ofNMDA-induced currents in the presence of SII. Changes in the peak NMDA currents determined during (initial effect) and after (late effect) the combined administration of NMDA with SII, SP + SII, or SP are expressed as the mean percentage of the control NMDA-induced current responses rf-SE for 6-I I cells.

we examined the responses to rapid applications of NMDA or NMDA plus SP in DH neurons perfused with a solution containing various concentrations of glycine (2 X loss10m6 M). This procedure was employed to allow preequilibration of glycine binding because, at nanomolar concentrations, diffusion-limited binding of glycine could significantly retard the interaction of glycine with NMDA receptors if a rapid changes in the concentrations of both glycine and NMDA were made simultaneously. The dependence of the SP enhancement of NMDA-induced current on extracellular glycine concentration was evaluated in a following manner. The effects of SP on the NMDA responses of DH neurons were compared by examining the same cells first under conditions of superfusion with glycine (5 X 1O-s-1O-6 M)-enriched solutions and then after the superfusing and drug solutions were exchanged with a nominally glycine-free solutions, which are likely to contain 120 nM glycine. This sequence of testing was chosen to rule out the possibility that a decrease of the SP enhancement of NMDA current usually seen with a second application of the same dose of peptide may be responsible for a modification of the NMDA responses of DH neurons to SP in the presence of glycine. Under these conditions, as shown in Fig. 10, the SP-induced potentiation of the NMDA-induced current, present when nominally glycine-free solutions were used (Fig. lOS), was absent in the presence of low7 M added glycine (Fig. 10, A and C). Moreover, in the presence of glycine concentrations of IO-‘- 1Oe6 M, a smah

reduction (to 79.0 & 5.5%, n = 15) in the initial peak NMDA current was observed both during (initial effect in Fig. 1OC) the coadministration of 2-5 nM SP plus 0. I mM NMDA and after (late effect in Fig. 1OC) removal of the peptide (to 86.1 t 2.8%, y1 = 10). In a few cells, a small increase during (by 113.0 + lO.O%, n = 3) and after (by 131.5 + 22.9%, n = 4) the removal of SP was recorded. Three cells were not affected by SP. As shown in Fig. 11, 7-chlorokynurenic acid, a competitive antagonist at the glycine regulatory site of the NMDA receptor channel complex, led to a reestablishment of the SP-potentiating effect. Thus, in the presence of glycine ( 10m7 M) and 7-chlorokynurenic acid (2 FM), 0.1 mM NMDA-induced current responses of DH neurons were enhanced by SP (2 nM) both during (by 120.3 -t 5.0%, n = 7) and after (by 129.3 + 5.4%, it = 8) removal of the peptide. In 2 cells, a small decrease ( -63%) was observed during coadministration of NMDA and SP.

Dependence of the SP efects on intracellular CaZf concentration Because it is known that SP can regulate [Ca”], (Rusin et al. 1991; Womack et al. 1988, 1989) and increase the voltage-activated Ca 2t channel currents in rat spinal DH neurons (Murase et al. 1989a; Ryu and RandiC 1990) and muscle cells (Mayer et al. 1990), the dependence of the SP-induced changes in the holding current, membrane


IS. I. RUSIN, P. D. RYU, AND M. RANDIC

274

A

B SP

(NMDA]

o-o 0-a

J 2-

l-

3-

Peak steady-state

4-

7 r-

f

0

ir

1 I a , tI -5

0

10

I,, , I 20

I, 1 I, I I, I 30

40

SP 20nM

C

I, I (

50

60 min

D

J 100PA 10s

-10

NKAl

10

nMO

20

min

6. N-methyl-D-aspartate (NMDA)-induced current responses in dorsal horn (DH) neurons are potentiated by substance P (SP) and neurokinin A (NKA). Traces show inward current responses evoked by 0.3 mM NMDA recorded at 2-min intervals from a DH neuron held at -50 mV before and after SP (20 nM for 1.5 min) application. First trace shows a control response to NMDA, whereas the 2nd, 3rd, and 4th traces are the responses recorded after the removal of SP at the times indicated on the graph (B). Whereas the peak inward component of NMDA-induced current was depressed initially (trace 2)) it was potentiated after the peptide removal (trace 3)) and the effect lasted almost 1 h. B: time courses of the peak and steady-state NMDA current responses recorded before and after SP administration are shown in the graph. Tetrodotoxin (TTX, 5 X 10e7 M) was present throughout. 1l-day-old rat. C: inward current responses evoked by 0.3 mM NMDA from a DH neuron held at -60 mV before and after NKA ( 1 nM for 5 min) application. The peak inward component of NMDA-induced current was depressed initially and potentiated thereafter, and the effect showed a recovery. D: time courses of the peak and steady-state NMDA current responses recorded before and after NKA administration. The perfusing solution contained 2 mM Ca2+, and the pipette solution contained 1 mM ethylene glycol-bis( P-aminoethyl ether)- N, N, N’, N’tetraacetic acid (EGTA, A and B). TTX (5 X 10s7 M) was present throughout. 13.day-old rat. FIG.

TABLE 2.

Modulation of NMDA-induced current after the pre-treatment of dorsal horn cellswith tachykinins

Types of Modulation Peak Initial effect Late effect Steady-state Initial effect Late effect

Neurokinin

Substance P (n = 93)

A (n = 15)

Increase

Decrease

Nil

Increase

Decrease

Nil

12 (126.6 t 6.1) 54 (146.6 t 6.8)

71 (64.8 t 2.1) 23 (70.4 t 3.1)

17

27 (133.0 +: 12.8) 73 (178.4 k 35.3)

53 (76.3 + 4.4) 0

20

8 (142.4 k 11.4) 9 (126.8 t 6.4)

9 (71.8 t 4.6) 7 (69.2 t, 5.6)

20 (112.0 ? 2.6) 33 (129.4 f: 14.0)

7 80 (one cell) 0

23 83 84

27 73 67

+ Values are percent of cells showing a particular type of interaction between NMDA and tachykinins; values in parentheses are percent change (means * SE) in the initial transient component (peak) and the steady-state component (steady-state) of NMDA-induced current immediately after (initial effect) and at the time of a maximal change (late effect) after removal of substance P or neurokinin A. NMDA, N-methyl-D-asparate.


MODULATION

250

3

t

150

200 0 -0 1 100 50 ‘~-0-o-o 0

275


.

2 I\ /

1

0

10

0

I’

0 .

I

0 O-0 \

0

4 o-o-o-o-~

I

o-0-0 I 50

1

I 30

1 20

\

v

1

I

I

40

min C

0

002

400+

0- 41 00

1 200 / oooo~o

I

0

\0 ‘ooo;~o

0

.

00

-10

I

10

20

SP

30 NM

00

0

5

0

0 \ 00 0 0

1 I

I1

I

40

50

60 min

FIG.7. &methyl-D-aspartate

(NMDA)-induced current responses in two Cs+-loaded DH neurons are potentiated by substance P (SP) and neurokinin A (NKA). A : traces show inward current responses evoked by 0.1 mM NMDA recorded at 2.5-min intervals at high speed on a digital oscilloscope diskette from a DH neuron held at -60 mV before (trace I ) and during 10-s application of 0.1 mM NMDA plus 2 nM SP (trace 2) or 0.1 mM NMDA plus 2 nM NKA (trace 5). B: time course of changes in the peak amplitude of the initial transient component of NMDA-induced current recorded before, during, and after SP + NMDA or NKA + NMDA coadministration in the same DH neuron. C: selective responses (as indicated in D) to 0.1 mM NMDA before (traces 1 and 3) and after SP (20 nM for 3 min; trace 2) or NKA (20 nM for 3 min; trace 4) are shown in the diskette traces. D: time courses of the peak (o ) and steady-state ( l ) NMDA current responses recorded before and after SP or NKA administration in the same DH neuron. TTX (5 X 10v7 M) was present throughout. The perfusing solution contained 0.5 mM Ca2+, and the pipette solution contained 140 mM CsCl and 1 mM ethylene glycol-bis( @-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). A-B: 13-day-old rat. C and D: 1 l-day-old rat.

current noise (Fig. 8), and NMDA-induced current on [ Ca2+li was studied by buffering the intracellular free Ca2+ concentration by 1-5 mM EGTA or I- 10 mM BAPTA (with O-O.5 mM added calcium). When EGTA concentra-

tion in the pipette solution was increased from 1 to 5 mM, we found that there was a tendency for a reduction of the SP enhancement of the NMDA-induced current after the pretreatment of DH cells with SP for l-5 min ( 1 mM EGTA:


276


A

SP 1 nM

J

100 pA IOpA

20 s

B

C

SP IO nM 1

1

SP 1 nM

FIG. 8. Substance P (SP) induces an inward current in isolated rat dorsal horn (DH) neurons that is accompanied with a change in membrane current noise. A and B: inward current responses evoked-by N-methyl-Daspartate (NMDA) in 2 different DH neurons voltage-clamped at -60 mV before and after SP ( 1 nM in A ; 10 nM in B) application. SP by itself induced an inward current accompanied by an increase (A and B) in membrane current noise and markedly reduced the peak inward component of the NMDA current response. C: SP alone.produced no change in the holding current, but it reduced membrane current noise and the peak inward component of NMDA-induced current. External solution (in mM): 150 NaCl, 5 KCl, 2 CaCl,, 10 D-&COSe, and 10 N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid (HEPES); 0.1 mg/ml BSA, pH 7.4. Pipette solution (in mM): 120 potassium aspartate, 20 KCl, 10 N&l, 1 MgCl*, 1 (A and C) or 5 (B) ethylene glycol-bis( ,8-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), 10 HEPES, 3 MgATP, 0.3 GTP, and 0.1 leupeptine, pH 7.2. Tetrodotoxin (TTX) (5 X 10m7 M) was present throughout. A and B: 13-day-old rats. C: 15-day-old rat.

NMDA-induced currents could result from a rise of [ Ca2’li (however, cf. Rusin et al. 1992). However, the holding potential (-60 mV) may have permitted activation of some low-threshold or transient component of the high-threshold calcium current (Ryu and RandiC 1990), which also could have contributed to changes of intracellular Ca2+. It is possible also that the inhibitory effects produced by BAPTA (Rusin et al. 1992) are not indicative of a role for Ca2+ as a second messenger in this case but of the necessity of having a basal [Ca2+]i high enough to allow effective G-protein function. To determine whether the SP effects were dependent on agonist-induced influx of extracellular Ca2+, the effects of SP were studied during bath perfusion of single cells (~1 = 5 1) with a NaCl-Ringer solution containing 0.2 mM Ca2+. Although lowering bath [Ca”] from 2.0 to 0.2 mM appeared to reduce the effects of SP pretreatment (4 min) on NMDA-induced currents, neither the inward shift nor the membrane current noise were significantly altered. Therefore the dependence of the SP actions on extracellular Ca2+ concentration still need to be evaluated on the same cells, first during control conditions and then after the perfusing solution is exchanged with a NaCl-Ringer solution containing either no added Ca 2 + and/or EGTA. In the present A

NMDA

0

170.4 t 24.0%, n = 8; 5 mM EGTA: 155.1 t 16.3%, n = 8). Because it has been known that EGTA often fails to effectively buffer transient increases in [ Ca2+Ii occurring near the cell membrane, we next used BAPTA as a calcium buffer because it binds Ca2+ more rapidly than EGTA (Tsien 1980). In five cells examined with the use of dialysis solutions that contained 10 mM BAPTA with nominally zero added calcium (the estimated free [ Ca2+li of this solution was - 10 -8 M; Chen et al. 1990), the SP-induced inward current and increase in membrane current noise (Fig. 8) were reduced or prevented. In addition, when the cells were pretreated for several minutes with SP, the SP-induced initial depression and late potentiation of the NMDA-induced current were noticeably different from those recorded with 1 mM EGTA or BAPTA-containing pipette solutions. As illustrated in Fig. 12, both the initial depressant effect and the late potentiation of the peak amplitude of the transient component of NMDA-induced current were reduced in four of five examined cells, and this effect is statistically significant (P < 0.05). These results are consistent with the idea that the SP effects on the holding current, membrane current noise (channel activity), and the

number

O-

0

of events

NMDA

peok HO\

-4

-3

-2

-1

0

2

3

5

7

8 min

FIG. 9. Substance P (SP)-induced enhancement of the transient component of iv-methyl-D-aspartate (NMDA)-induced current is accompanied by an increase in the frequency of brief inward current steps. Traces show inward current responses evoked by 0.1 mM NMDA recorded at 2.5-min intervals from a DH neuron held at -60 mV before, during, and after 2 nM SP application (A ). The peak increase of the initial transient component of NMDA-induced current occurred 3 min after exposure of a cell to SP, and it coincided with the maximum increase in the frequency of inward current steps (B). The perfusing solution contained 2 mM Ca2+ and 5 X 1Om7M tetrodotoxin (TTX); the pipette solution contained 1 mM ethylene glycol-bis( ,&aminoethyl ether)-N,N,N’,N’-tetraacetic acid ( EGTA ) . 13-day-old rat.


MODULATION

A SP2nM [NMDA]

-

3 min

277


0-0

5 min

initial -0

=

late

effect effect

-@ E8 160

GiY

8 min

I

20 pA

10s

85 -8 Log

-7 [glycine]

(M)

FIG. 10. Interaction of glycine with the substance P (SP) enhancement of N-methyl-D-aspartate (NMDA)-induced currents. Inward current responses induced by 0.1 mM NMDA recorded before, during, and after the combined administration with SP in the presence (A) or absence (B) of glycine. A : in the presence of glycine (20 nM), the peak inward component of NMDA-induced current was reduced to a small degree during the coadministration of SP + NMDA and changed little after the removal of the peptide. B: peak inward NMDA-induced current, recorded in a nominally 0-glycine-containing solution, was increased by SP (2 nM) both during and after the peptide removal. Holding potential -60 mV. The perfusing solution contained 0.5 mM Ca2+, and the pipette solution 1 mM bis-( o-aminophenoxy)-N,N,N’,N’-tetraacetic acid (BAPTA). 14-day-old rat. C: interaction of glycine with the SP potentiation of NMDA-induced currents. Changes in the peak component of NMDA-induced currents measured during (initial effect) and after (late effect) the combined administration of NMDA with SP in the presence of IO-‘- 10v7 M glycine were plotted. Data are expressed as means + SE from determinations for 5-9 cells. Tetrodotoxin (TTX, 5 X 10e7 M) was present throughout.

experiments, this was not done, because the conditions of the cell deteriorated rapidly when nominally zero-calcium solutions were used. Phorbol esters and forskolin mimic the eficts of tachykinins on the NMDA-induced current response To examine the molecular mechanisms underlying the changes in NMDA-induced currents caused by tachykinins, we examined the effects of phorbol esters, because it has been reported that the spinal DH contains high levels of binding sites for phorbol esters (Mantyh et al. 1984) and that protein kinase C (PIE) is present in the rat spinal DH ( Mochly-Rosen et al. 1987). Because PKC activation can be mediated directly by phorbol esters, in the absence of phosphoinositide breakdown, we used these agents to examine the effects of the enzyme activation on the chemical sensitivity of NMDA receptors of freshly isolated rat spinal DH neurons to a specific agonist. When single DH neurons (n = 15) were exposed to PDBu ( 10-7- 1Ob6 M) for 2-8 min before the testing of NMDA responses, a marked and long-lasting increase in the peak amplitude of the transient component of NMDAinduced current was observed in 9 of 15 examined cells (Fig. 13 B). In another three cells, the potentiation was preceded by a depression of the NMDA response. For comparative reasons, three cells were tested with 4cu-PDiDec, a phorbol analog unable to activate PKC. Although 4cu-PDiDee ( 10e7 M) did not induce any significant change in the NMDA response, the NMDA-induced currents consistently increased in the same cells after addition of active phorbol esters.

Although, in the present work, phorbol esters augmented the NMDA-induced current responses of single DH neurons to NMDA, the identity of endogenous substance(s) participating in this effect is presently unknown. The fact that PKC has nonneuronal localization in the rat spinal DH ( Mochly-Rosen et al. 1987) makes its direct involvement less likely. There are many reports indicating that phorbol esters have effects on adenosine 3 ‘,5 ‘-cyclic monophosphate (CAMP) metabolism in mammalian tissues (Bell et al. 1985 ). It is therefore of interest that when we observed in nine cells (n = 12 ) exposed to forskolin ( 10 -6-2 X 1OA5 M for 2-8 min), an adenylate cyclase activator, the modulation of NMDA response consisting of either an initial depression followed by a potentiation (Fig. 13A, n = 4) or potentiation alone ( n = 5). Moreover, we have recently shown that staurosporine (5 X 10-8-10-7 M for 5 min), the agent known to inhibit both protein kinase C- and CAMP-dependent protein kinase, reduced the SP- and NKA-caused enhancement of NMDA-induced current (both percent increase and duration of the effect) in DH cells (Fig. 13C). This observation suggests that phosphorylation of the NMDA receptor-ion channel complex, or a related protein, by a staurosporinesensitive protein kinase( s) may be involved in the mediation of the tachykinin effects. Modulation of QA- and AMPA-induced currents by SP To investigate the effects of SP on the QA- and AMPAinduced currents, we perfused a cell with SP ( 2 X 10 -lo-2 X 10e6 M) for 30 s-2.5 min before starting the applications of QA or AMPA at 2-min intervals for 260 min. In the major-


278


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RG. 11. Interaction of glycine with the substance P (SP) enhancement of N-methyl-D-aspartate (NMDA)-induced current-effect of 7-chlorokynurenic acid. Inward current responses induced by 0.1 mM NMDA recorded in 2 different DH neurons (a and b) before, during, and after the combined administration with 7-chlorokynurenic acid (7ClKyn, A) or SP in the presence of glycine (Gly, 0.1 PM, B) or gly + 7ClKyn (B) in the perfusing solution. A: 7-Chlorokynurenic acid (2 PM), a competitive antagonist at the glycine allosteric site of the NMDA receptor channel complex, reversibly blocked the peak inward component of NMDA-induced current and significantly reduced the steady-state component (middle). B: development of the SP enhancement of NMDA-induced currents is prevented in the presence of 0.1 pM glycine in the perfusing solution (top). 7-Chlorokynurenic acid, applied with glycine in the perfusing solution, led to a reestablishment of the SP enhancement of NMDA response (bottom). Holding potential -60 mV. The perfusing solution contained 0.5 mM Ca2+, and the pipette solution contained 5 mM EGTA. C: effects of the presence of glycine and 7-chlorokynurenic acid in perfusing solution on the SP enhancement of NMDA-induced currents. Changes in the peak component ofNMDA-induced currents measured during (initial effect) and after (late effect) the combined administration with 2 nM SP + 0. I uM Glv or 1 pM SP + 0.1 PM Gly or 2 nM SP + 0.1 PM Gly + 2 PM 7ClKyn or 2 nM SP. Data are expressed as means + SE’from determinations for 6-10 cells. Tetrodotoxin (TTX) is present throughout.

ity of the experiments, 5 X lo-’ M TTX was present in the perfusing solution. The currents activated by QA at concentrations of 1Oe6 M ( Ascher and Nowak 1988a), our use of 1 mM external Mg2+ in the


MODULATION

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marked potentiation, the AMPA-induced current appears to be only potentiated. The SP-potentiating effect was prevented when internal Ca2+ was buffered with 10 mM BAPTA and external solution contained 2 mM Ca2+ (II = 4). KA (5-50 PM)-induced inward currents appear to be least affected by tachykinins (Fig. 14 B) . SP (2- 100 nM for l-4 min) increased (by 127%) the amplitude of the KA-induced current only in 2 of 23 examined cells and decreased it (to 9 1% ) in 5 cells. NKA (2- 100 nM for 2-5 min) increased (by 116%) the KA response in two cells and decreased (to 74%) in another two cells.

A (NMDA]

Fotskolin )

-

12. Dependence of the substance P (SP) enhancement of Nmethyl-r>-aspartate (NMDA)-induced current on intracellular free Ca2+ concentration. In the graph, the x axis shows concentration of ethylene glycol-bis( @-aminoethyl ether) - N, N, N’, N’-tetraacetic acid ( EGTA, 1 or 5 mM ) and bis- ( o-aminophenoxy ) -N, N, N’, N’-tetraacetic acid (BAPTA, 10 mM) in the pipette solution, and the y axis shows change in the peak amplitude of NMDA-induced current produced by SP (2 nM) relative to control value ( expressed as 100% ) . Open circles: changes in the peak component of NMDA-induced current measured for the first response (initial effect) recorded after removal of SP; closed circles: changes measured at a maximum potentiation (late effect) produced by SP. Data are expressed as means t SE from determinations for 3-l 1 cells.

1 Om5M

JlrT T

FIG.

perfusing solution tends to exclude this possibility. Besides ionotropic AMPA receptors, metabotropic glutamate receptors are also activated by QA (Sugiyama et al. 1987). It will be of interest, therefore, in a future work to examine the possibility of interaction of SP with lS3R=ACPD, a highly selective agonist at the metabotropic receptor (Schoepp et al. 199 1) . Trans.ACPD is known to produce a slow depolarization of CA, hippocampal neurons (Stratton et al. 1989, 1990), neurons in the dorsolateral septal nucleus of the rat (Zheng and Gallagher 199 1 ), and substantia gelatinosa cells (unpublished observation). Although a highly potent excitant acting in submicromolar concentrations at the non-NMDA receptors, QA also shows high affinity for other EAA receptors, such as the KA and metabotropic receptors. A structural analogue of QA, AMPA, is more specific for the non-NMDA subtype of EAA receptors in the mammalian CNS, and it has little affinity for 3H-KA binding sites (Watkins et al. 199 1). To investigate whether tachykinins besides QA-induced current may also modulate AMPA-induced current responses of freshly isolated rat DH neurons, we examined the effects of SP and NKA on the AMPA-induced inward currents. Pretreatment with SP (0.5-10 nM for l-5 min) enhanced the amplitude of the AMPA-induced current in 8 of 19 tested cells (by 118.3 t 4.0%), whereas NKA ( lo-20 nM for 30 s-2.5 min) increased it (by 134.0 t 12.2%) in 3 of 4 examined cells. In one of 19 cells, a decrease (to -70%) of the AMPA-induced current was observed after administration of SP. The enhancing effects of SP and NKA on both the AMPA- and the NMDA-induced current responses in the same cell are illustrated in Fig. 15. In difference to the behavior of the NMDA responses where NKA produced an initial transient depression followed by a

13 min

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13. Possible participation of postsynaptic protein kinases activity in the modulation of N-methyl-D-aspartate ( NMDA )-induced currents in isolated dorsal horn (DH) neurons by tachykinins. Traces show inward current responses induced by 0.1 mM NMDA recorded at 2.5min intervals from 2 different DH neurons held at -60 mV before and after forskolin (A) or PDBu (B) application. First trace shows a control response to NMDA, whereas the second, third, and fourth traces are the responses recorded after the removal of forskolin (A) or PDBu (B) at the times indicated. Whereas the peak inward component of NMDA-induced current was depressed initially (trace 2)) it was potentiated 7 min after the removal of forskolin ( 10 PM for 2 min) and 13 min after the removal of 4@-phorbol- 12,13-dibutyrate (PDBu, 0.1 PM for 2 min). The perfusing solution contained 0.5 mM Ca2+, and the pipette solution contained 1 mM ethylene glycol-bis( ,&aminoethyl ether) - N,N, N’,N’-tetraacetic acid (EGTA). Tetrodotoxin (TTX, 5 X 10e7 M) was present throughout. A: 12-day-old rat. B: lo-day-old rat. C: time course of the peak NMDA current responses recorded before, in the presence of, and after administration of 50 nM staurosporine (SS) + 10 nM substance P (SP) or 10 nM SP alone. The DH neuron was exposed to staurosporine for 3 min before the coadministration of SS + SP (2 min). TTX was present throughout. The perfusing solution contained 0.5 mM Ca2+, and the pipette solution contained 140 mM CsCl and 1 mM EGTA. FIG.



280

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14. Selective enhancement of quisqualate (QA)-induced current by substance P (SP). A : inward current responses evoked by 5 PM QA recorded at 2-min intervals from a DH neuron, held at -60 mV, before, during, and after 20 or 100 nM SP administration. Both the transient and steady-state components of QA-induced current were augmented by SP in a dose-dependent manner, and the effect showed little recovery 24 min after removal of the peptide. The perfusing solution contained 0.2 mM Ca*+, and the potassium aspartate pipette solution contained 1 mM bis-(o-aminophenoxy)-N,N,N’,N’tetraacetic acid (BAPTA). Tetrodotoxin (TTX, 5 X lo-’ M) was present throughout. 12-day-old rat. B: inward current responses evoked by 5 PM QA or 10 PM kainate (KA) recorded alternatively at 2-min intervals from the same DH neuron, held at -60 mV, before, during, and after 200 nM SP administration. The responses to QA recorded at 90 s, 10 min, and 13 min after SP, and to KA at 3,8, and 11 min after SP. Whereas both components (transient and steady-state) of QA-induced current were enhanced by SP, KA-induced current in this cell was not affected. The external solution contained 2 mM Ca, and the pipette solution contained 1 mM BAPTA. 13-day-old rat. Cand D: incidence of dorsal horn (DH) cells showing the increase of the initial peak (C) and steady-state (D) components of QA-induced current after pretreatment with SP was to a large extent dependent on the concentration of QA used. FIG.

DISCUSSION

Tachykinin-induced current

potentiation

of NMDA-induced

In the present investigation, we have shown that tachykinins, SP, and NKA, modulate NMDA- and QA/AMPAinduced current responses of a proportion of freshly isolated spinal DH neurons from young rats. For a given tachykinin concentration, SP and NKA were most effective in producing an increased NMDA receptor-mediated current response, both in the amplitude of the peak currents generated and in the percentage of cells that responded to these peptides. The effects were observed at tachykinin concentrations as low as IO-’ ’ M in an external solution containing nominally zero glycine. This effective

tachykinin concentration was significantly lower than that of lo-’ M observed to enhance the NMDA responses ofthe rat DH neurons studied in a slice preparation (unpublished observations). However, a strong potentiation of the behavioral responses in mice was recently seen when picomolar amounts of SP were coadministered with NMDA, AMPA, and KA (Mjellem-Joly et al. 1991). The SP potentiation of NMDA-induced current in the present work was blocked by a claimed novel nonselective antagonist of neurokinin receptors, spantide II (Folkers et al. 1990; Rusin and RandiC 199 1)) suggesting that the SP effect is a true tachykinin receptor-mediated event. On the basis of the similar enhancing effects observed with SP and NKA, the interactions between tachykinins and NMDA receptors appear to be mediated by both NK-1 and NK-2 receptors.


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Possiblecellular mechanisms qf the SP enhancement of NA4DA response The exact molecular mechanisms underlying the enhancement of NMDA receptor-activated conductance by tachykinins have yet to be elucidated. In the presence of high ( 1 PM) external glycine, the possibility that SP directly modifies kinetic properties of single NMDA channels, or unmasks silent NMDA receptor channel complexes, seems unlikely because the NMDA-activated single-channel currents recorded in the outside-out configuration of the patch-clamp method in excised patches of cultured cerebral cortical neurons were not significantly modified by SP (RandiC et al. 1990). Alternatively, SP may act at one or more regulatory sites known to be associated with the NMDA receptor-ion channel complex (Kemp et al. 1987). The best-characterized modulation of the NMDA receptor activity is by glycine (Johnson and Ascher 1987). Glycine is known to enhance the activity of NMDA channels, and the strychnine-resistant, high-affinity glycine binding site is located at the NMDA receptor-ion channel complex (Johnson and Ascher 1987; Kushner et al. 1988). Glycine is presently thought to act either as a coagonist (Kleckner and Dingledine 1988) or a regulator of the rate of desensitization at NMDA receptors (Benveniste et al. 1990; Mayer et al. 1989; Vyklicky et al. 1990). Glycine enhances both the initial transient and steady-state compo-

FIG. 15. Substance P (SP) and neurokinin A (NKA) enhance N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) responses in same DH neurons. A: inward current responses evoked by 0.1 mM NMDA (lop) or 10 PM AMPA (bottom) recorded at I-min intervals before and after SP administration. In each row, truce I represents a control response, whereas truces 2-4 show the responses to NMDA or AMPA recorded after SP application ( 1 nM for 75 s, trace 2; I nM for 4 mitt, truces 3 and 4) at the times indicated in the graph (B). The NMDA-induced current was depressed initially and potentiated thereafter (B). The AMPA-induced current was augmented by SP, and this effect showed no recovcry. B: time course of the peak NMDA and AMPA current responses recorded before and after SP administration. The external solution contained 2 mM Cal+, and the pipette solution contained 5 mM ethylene glycol-bis(Paminoethyl ether)- N,N,N’.N’-tetraacetic acid (EGTA). Tetrodotoxin (TTX. 5 X IO-’ M) was present throughout. Holding potential -60 mV, 15-day-old rat. C: inward current responses evoked by 0.3 mM NMDA (top) or 10 WM AMPA (bottom) from a DH neuron held at -60 mV, before and after NKA (10 nM for 2 min) administration. Both NMDAand AMPA-Induced currents were potentiated by NKA. The external solution contained 2 mM Ca’+ and 5 x IO-’ M TTX: the pipette solution contained I mM EGTA. I3day-old rat.

nents of NMDA-induced current (starting at glycine concentrations of -1 nM) in freshly isolated rat spinal DH neurons (Murase et al. 1989b, 1990). We report here that 5 X 1O-*- 1Oe6 M glycine in the perfusing solution reduced or abolished the SP-induced potentiation of the NMDA response (Fig. 1OC) observed when nominally glycine-free EAA and/or peptide solutions, which are likely to contain >20 nM glycine, were used. Moreover, 7-chlorokynurenic acid, a competitive antagonist at the glycine regulatory site of the NMDA receptor-channel complex, led to the reinstitution of the SP-potentiating effect (Fig. 11 B). Although molecular mechanism(s) underlying the dependence of the SP-induced enhancement of the NMDA receptor-activated conductance on external glycine concentration is presently unknown, the possible role of Ca2+ ions and their interactions with glycine allosteric mechanism needs to be examined in a future. In this context, three recent findings are of interest. First, we found (Rusin et al. 199 1) that in the presence of 100 nM added glycine in the external medium there was no potentiation of the NMDA-dependent increases in [Ca2+], in DH neurons. Second, Gu and Huang ( 199 1) observed that the potentiation of NMDA responses of the rat trigeminal neurons by Ca2+ ions depended on external glycine concentration. Thus, in low external glycine, the NMDA-activated currents were increased as the extracellular free Ca2+ concentration


282


( [Ca2+],) was raised from 2 to 20 mM, whereas in high there is a possibility that this process may play an important external glycine, the NMDA response was decreased. They role in synaptic plasticity. suggested that Ca 2+ ions potentiate the NMDA receptorThe intracellular pathway linking SP receptor activation activated conductance by increasing the affinity of glycine to increases in the [ Ca2+li is presently uncertain, but may to NMDA receptor channel complex. Third, O’Malley et involve formation of phosphoinositide metabolites (Manal. ( 199 1) reported that SP (and C terminal fragments of tyh et al. 1984; Watson and Downes 1983). In addition, we SP) in concentrations of 0. l- 100 PM did not have affinity have demonstrated that perfusion of rat spinal slices with for or change the kinetics of [ 3H] glycine binding. Instead, phorbol esters, the agents known to activate the calciumSP markedly enhanced [ 3H] MK-80 1 binding. and phospholipid-dependent protein kinase C (PKC; NiAnother, perhaps even more likely way in which the acti- shizuka 1984, 1988)) selectively enhanced the depolarizing vation of SP receptors may modify glutamate and NMDA responses of DH neurons to NMDA and L-glutamate receptor-activated conductances of DH neurons is indi(Gerber et al. 1989). Similar potentiation of the NMDArectly through the regulation of intracellular mechanisms induced currents by active phorbol esters was seen in the (Mantyh et al. 1984). Our finding that the SP enhancement present work when freshly isolated DH neurons were used of NMDA-induced current was reduced when [ Ca2+li was (Fig. 13 B). However, the finding that PKC has non-neubuffered with 10 mM BAPTA suggested that the tachy- ronal localization in the rat spinal DH (Mochly-Rosen et kinin-induced changes in [ Ca2+li may play a role. al. 1987) makes its direct involvement less likely. In addiOur results established that NMDA, non-NMDA, and tion, DH apparently lacks IP, receptors (Worley et al. tachykinin (SP, NKA) receptors are coexpressed on single 1987), raising the possibility that other second messenger DH neurons. Furthermore, it is known that there is a con- systems may contribute to the SP-induced increases in vergent regulation of [ Ca2Q in rat DH neurons by SP (Wo[ Ca2+li levels. It is known that activation of PKC can enmack et al. 1988, 1989) and glutamate receptor agonists hance the accumulation of CAMP (Bell et al. 1985 ) and that (MacDermott et al. 1986; Mayer et al. 1987; Mayer and the superficial spinal DH contains high levels of binding Miller 199 1). SP increases [ Ca2Q in a proportion of sites for forskolin. The possibility that the activation of adeacutely dissociated rat DH neurons via two distinct mechanylate cyclase-AMP-dependent protein kinase system may nisms: 1) by promoting Ca2+ entry through voltage-depenbe involved in the regulation of the activity of NMDA redent Ca2+ channels (Rusin et al. 199 1, 1992; Ryu and Ranceptors in rat spinal DH neurons is supported by our recent die 1990) and 2) by mobilizing intracellular Ca2+ stores results. We found that forskolin, a potent activator of ade(Womack et al. 1988, 1989). The increase in [Ca2+li after nylate cyclase, and membrane permeant analogs of CAMP glutamate application appears to result from Ca2+ entry enhance the sensitivity of NMDA responses of the rat DH through voltage-dependent Ca2+ channels activated by cell neurons in a slice preparation (Cerne et al. 1989; Gerber et depolarization or via NMDA-gated channels ( MacDermott al. 1989) and also in freshly isolated rat spinal DH neurons et al. 1986; Mayer and Miller 1991; Murphy et al. 1987). (Fig. 13A). These results suggest the possibility that the The hypothesis that the SP-induced enhancement of the alteration in [ Ca2+li and consequential changes in at least responses of acutely isolated rat spinal DH neurons to two second messenger systems may be involved in the reguNMDA (Randic et al. 1990) may be in some way related to lation of the activity of neuronal NMDA receptors. the changes in [Ca2+li is supported by our recent finding Moreover, we have shown in this work that staurosporthat tachykinins potentiate the glutamate- and NMDAine, the agent known to inhibit both PKC and CAMP-deevoked increases in [ Ca2Q measured using fura- based pendent protein kinase, reduced the SP-caused enhancemicrofluorimetry (Rusin et al. 199 1, 1992). Thus this con- ment of NMDA-induced current. This finding suggests that vergent regulation of [ Ca2+li by EAA and tachykinins may phosphorylation of the NMDA receptor-ion channel comprovide a cellular mechanism underlying the interactions plex, or a related protein, by a staurosporine-sensitive probetween NMDA and tachykinins. tein .kinase( s) may be involved in the mediation of the Although the exact mechanism of action of Ca2+ is un- tachykinin effects. known, changes in [ Ca2+li can lead to activation of Ca2+dependent protein kinases or phosphatase enzymes, result- Possible functional implications of the tachykinin- ,induced ing in changes in the concentration of several second mes- potentiation of excitatory amino acid responses sengers and protein phosphorylation. Recent observations are consistent with the hypothesis that phosphorylation of Evidence indicates that the NMDA receptor mechanism, the NMDA channel itself, or a related protein, appears to be besides playing an important role in normal signal processrequired to maintain functional state of the channel (Bading (Gerber and Randic 1989; Gerber et al. 199 1; Kangrga ing and Greenberg 199 1; MacDonald et al. 1989). There is et Bi. 1988), is also involved in the mediation of various a direct evidence that phosphorylation of other ligandforms of neuronal pl .asticity, such as use-dependent modifigated ion channels such as nicotinic ACh (Huganir et al. cations of synaptic gain in the adult nervous system. Long1986) and y-aminobutyric acid (GABA), receptors term potentiation (LTP) in area CA, of the hippocampus is (Porter et al. 1990) regulates their function. Phosphorylathe most extensively studied form of activity-dependent tion.of nicotinic ACh receptor is catalyzed by at least three synaptic plasticity in the vertebrate nervous system (Akers different protein kinases (protein kinase C, CAMP-depenet al. 1986; Collingridge and Singer 199 1; Mayer and Miller dent protein kinase and tyrosine-specific protein kinase) 199 1). A role of NMDA receptors in the induction of LTP (Miles and Huganir 1988 ) . Although the functional signifiis well established. Pharmacological studies have suggested cance of phosphorylation of the receptors has been unclear, that PKC is essential for maintenance of LTP, but it is not Downloaded from www.physiology.org/journal/jn by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 17, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

MODULATION


known which receptors, when stimulated, lead to activation of this enzyme. Although a great deal is known about LTP in hippocampus, the existence of a similar synaptic plasticity at primary afferent synapses in the rat spinal DH has been only recently demonstrated (Cerne et al. 199 1) . However, the molecular mechanisms underlying this phenomenon have yet to be elucidated. Our results suggest that besides CGRP ( Ryu et al. 1988 ) , and a CL-opioid receptor (Rusin and Randic 199 la), the tachykinin receptors may also be involved in this process ( Rand2 et al. 1990). Tachykinin (Mantyh et al. 1984) and NMDA (Greenamyre et al. 1984; Monaghan et al. 1989) receptors are present in high densities in the superficial spinal DH. In a number of model systems, SP receptors appear to signal their actions by eliciting breakdown of inositol phospholipids (Mantyh et al. 1984), leading to the production of inositoltrisphosphate (IP,) and diacylglycerol (DAG) . IP, releases Ca2+ from intracellular stores, whereas DAG activates PKC. When activated by DAG, the C kinase phosphorylates specific substrate proteins that contribute to various cellular processes, including neurotransmitter release and receptor-transducing mechanisms (Berridge 1986; Berridge and Irvine 1984; Nishizuka 1984). It is well established that many forms of PKC are regulated by [ Ca2+li. Thus potentiation of NMDA-induced increase in [Ca2+]i by tachykinins in acutely isolated rat spinal DH neurons (Rusin et al. 199 1) may synergize with activated PKC in some way to produce the changes in postsynaptic sensitivity to glutamate currently thought to be responsible for LTP. We have recently shown that in the rat spinal DH, tachykinins (SP, NKA) and phorbol esters potentiate the basal and dorsal root stimulation-evoked release of endogenous glutamate and aspartate (Gerber et al. 1989; Kangrga and Randic 1990) and also enhance the sensitivity of postsynaptic NMDA receptors (Gerber et al. 1989; Hecimovic et al. 1990; Rand% et al. 1990). These interactions between EAAs and tachykinins occurring both at presynaptic and postsynaptic sites may serve the purpose of regulating the capacity for sensory information transfer at the first CNS synaptic relay at which the initial processing and integration of cutaneous information, including pain, takes place.

Potential sign$cance of EM /peptide interactions for hyperalgesia After injury to nervous tissue, the nociceptive elements undergo dynamic changes, but the cellular mechanisms underlying hyperalgesia and neuronal plasticity in nociceptive systems are not well understood. Hardy et al. ( 1967 ) distinguished primary hyperalgesia that occurs at the site of injury and secondary hyperalgesia, which develops over several minutes in the surrounding area and lasts for hours. A role for sensitization of nociceptors in the generation of primary hyperalgesia is well documented ( LaMotte and de Lanerolle 1983; Lynn 1979; Meyer and Campbell 198 1). More recent work has begun to define a role of an increased excitability of central neurons, including spinal DH neurons, in the generation of secondary hyperalgesia (Woolf and Thompson 199 1). One possible hypothesis is that a mechanism involving synaptic co-release of EAA and peptides and increased responsiveness of DH neurons on noxious stimulation may underlie the development of sen-

283

sitization of DH neurons and contribute, at least in part, to secondary hyperalgesia. A model has been proposed whereby neuropeptides [ SP, calcitonin gene-related peptide (CGRP), dynorphin, and Tyr-D-Ala-Gly-Me-Phe-Glyol-enkephalin (DAGO)] enhance excitability at NMDA receptor sites, leading first to DH neuronal hyperexcitability and then to excessive depolarization and excitotoxicity. The latter may lead to loss of inhibitory neurons. The combined effects of depolarization and loss of inhibition would contribute to the hyperexcitability and expansion of receptive fields and lead to behavioral hyperalgesia (Dubner 1991). EAA and tachykinins provide likely candidates for the participation in the development of sensitization of DH neurons, because they are known to coexist in small primary sensory neurons that are likely to be nociceptive (DeBiasi and Rustioni 1988 ) . Painful stimuli cause release of EAA (Kangrga and Rand2 1990, 199 1; Skilling et al. 1988) and SP (Yaksh et al. 1980) at the terminals of nociceptive primary afferents in the spinal DH. We have recently demonstrated that SP increases the basal and depolarizationevoked release of glutamate and aspartate from the rat spinal DH slice (Kangrga and Rand2 1990) and, as shown in the present work, tachykinins cause also a long-lasting enhancement of the responses of a proportion of freshly isolated spinal DH neurons to glutamate (Randic et al. 1990), NMDA, AMPA, and QA. In addition, Dougherty and Willis ( 199 1) demonstrated increased responsiveness of primate spinothalamic (STT) neurons after the combined administration of EAA and SP. The potentiating effect of SP lasted for hours and was accompanied by an increase in the responses of the STT cells to mechanical stimulation of skin, thereby providing a cellular model of secondary hyperalgesia (Hardy et al. 1967). Participation of EAAs and SP in the generation of a long-lasting potentiation of various nociceptive reflexes ( Mjellem-Joly et al. 199 1) and in the development of prolonged increase of spinal cord excitability after repetitive stimulation of C-afferents has also been demonstrated ( Wiesenfeld-Hallin et al. 199 1; Woolf and Wiesenfeld-Hallin 1986). Thus the results of electrophysiological and behavioral experiments indicate a functional interaction between SP and glutamate in the mammalian spinal DH compatible with the hypothesis that co-release of SP and glutamate from primary afferent neurons may enhance nociception. Considerable evidence supports a role for glutamate and/ or aspartate in the fast excitatory synaptic transmission from both large- and small-diameter primary afferent fibers, including nociceptive afferents (Gerber and Randic 1989; Kangrga and Rand% 1990, 199 1; Yoshimura and Jesse111990). The present view is that the activation of the non-NMDA receptors is involved in the transmission of low-threshold signals, whereas NMDA receptors participate in the transmission of high-threshold (nociceptive) responses (Dickenson 1990). Small-diameter primary afferents, in contrast to large myelinated fibers, have the capacity to generate besides fast excitatory synaptic potentials, slow excitatory synaptic potentials in spinal DH neurons that may involve EAA and SP (Gerber and Randic 1989; Gerber et al. 199 1; Urban and Randic 1984). These slow potentials can summate to produce large depolarizations


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that outlast the stimulus for several minutes and in this way could recruit subliminal inputs to a suprathreshold level. These relatively short changes cannot alone explain prolonged receptive field alterations and hyperalgesia. A plausible working hypothesis would be that the substances involved in the generation of slow potentials such as SP (Gerber et al. 199 1; Murase and Rand2 1984; Urban and Randic 1984), and the activation of NMDA receptors (Gerber and Randic 1989; Kangrga et al. 1988; Monaghan et al. 1989; Woolf and Thompson 199 1) may trigger longer-lasting changes through alterations of the level of [Ca2+li (Rusin et al. 1991; Womack et al. 1988, 1989) and activation of second messenger systems, which in turn causes a sustained change in membrane properties (Gerber et al. 1989). Afferent inputs have been shown to alter [ Ca2+]i levels (Rusin et al. 199 1; Womack et al. 1988, 1989)) PKC activity, and the expression of c- fos proto-oncogene (Hunt et al. 1987 ) in DH neurons. We thank Dr. H. HeCimoviC for his help in some experiments. This research was supported in part by National Institute for Neurological and Communicative Disorders and Stroke Grant NS-26352 and Grant BNS 84 18042 from the National Science Foundation. Present addresses: K. I. Rusin, Department of Physiology, the University of Michigan Medical School, Ann Arbor, MI 48 109-0622; M. Randic, Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 500 11; P. D. Ryu, Department of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, Suwon 44 1 744, Republic of Korea. Permanent address of K. I. Rusin: Department of Spinal Cord Physiology, A. A. Bogomoletz Institute of Physiology, Kiev, GSP 252 60 1, USSR. Correspondence should be addressed to M. Randic at the above address. Received 12 September 199 1; accepted in final form 10 March 1992. REFERENCES G. IS. AND RASMUSSEN, K. Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse 3: 331-338, 1989. AKERS, R. F., LOVINGER, D. M., COLLEY, P. A., LINDEN, D. J., AND ROUTTENBERG, A. Translocation of protein kinase C activity may mediate hippocampal long-term potentiation. Science Wash. DC 23 1: 587589, 1986. ASCHER, P. AND NOWAK, L. Quisqualate- and kainate-activated channels in mouse central neurones in culture. J. PhysioZ. Lond. 399: 227-245, 1988a. ASCHER, P. AND NOWAK, L. The role of divalent cations in the N-methylD-aspartate responses of mouse central neurones in culture. J. Physiol. Land. 399: 247-266, 1988b. BADING, H. AND GREENBERG, M. E. Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science Wash. DC 253: 912-914, 1991. BELL, J. D., BUXTON, I. L. O., AND BRUNTON, L. L. Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha S-GTP-catalytic subunit interaction. J. Biol. Chem. 260: 2625-2628, 1985. BENVENISTE, M., CLEMENTS, J., VYIUICK~, L., JR., AND MAYER, M. L. A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones. J. Physiol. Lond. 428: 333-357, 1990. BERRIDGE, M. Second messenger dualism in neuromodulation and memory. Nature Lond. 323: 294-295, 1986. BERRIDGE, M. J. AND IRVINE, R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature Lond. 3 12: 3 15-32 1, 1984. BROWNING, M. D., BUREAU, M., DUDEK, E. M., AND OLSEN, R. W. Protein kinase C and CAMP-dependent protein kinase phosphorylate the B subunit of the purified y-aminobutyric acid A receptor. Proc. Natl. Acad. Sci. USA 87: 13 15-l 3 18, 1990. AGHAJANIAN,

R., GERBER, G., KANGRGA, I., AND RANDI~, M. Effects of cyclic3 ‘,5 ‘-adenosine monophosphate on rat spinal dorsal horn neurons in vitro. Sot. Neurosci. Abstr. 15: 1320, 1989. CERNE, R., JIANG, M. C., AND RANDI~, M. Long-lasting modification in synaptic efficacy at primary afferent synapses with neurons in rat superficial spinal dorsal horn. Sot. Neurosci. Abstr. 17: 133 1, 199 1. CHARPAK, S. AND G;~HWILER, B. H. Glutamate mediates a slow synaptic response in hippocampal slice cultures. Proc. R. Sot. Land. B BioZ. Sci. 243: 221-226, 1991. CHEN, Q.-X., STELZER, A., KAY, A. R., AND WONG, R. K. S. GABA, receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones. J. Physiol. Lond. 420: 207-22 1, 1990. COLLINGRIDGE, G. L. AND SINGER, W. Excitatory amino acid receptors and synaptic plasticity. In: The Pharmacology of Excitatory Amino Acids. A SpeciaZ Report, Edited by D. Lodge, and G. Collingridge. Cambridge: Elsevier, 199 1, p. 42-48. COTMAN, C. W. AND IVERSEN, L. L. Excitatory amino acids in the brainfocus on NMDA receptors. Trends Neurosci. 10: 263-265, 1987. DEBIASI, S. AND RUSTIONI, A. Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord. Proc. NatZ. Acad. Sci. USA 85: 7820-7824, 1988. DICKENSON, A. H. A cure for wind up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307-309, 1990. DOUGHERTY, P. M. AND WILLIS, W. D. Enhancement of spinothalamic neuron responses to chemical and mechanical stimuli following combined micro-iontophoretic application of NMDA and substance P. Pain 47: 85-93, 1991. DUBNER, R. Neuronal plasticity and pain following peripheral tissue inflammation or nerve injury. In: Proceedings of the VIth World Congress on Pain, edited by M. R. Bond, J. E. Charlton, and C. J. Woolf. Amsterdam: Elsevier, 199 1, p. 263-276. FENWICK, E. M., MARTY, A., AND NEHER, E. Sodium and calcium channels in bovine chromaffin cells. J. Physiol. Lond. 33 1: 599-635, 1982. FOLKERS, K., FENG, D.-M., ASANO, N., H~ANSON, R., WIESENFELDHALLIN, Z., AND LEANDER, S. Spantide II, an effective tachykinin antagonist having high potency and negligible neurotoxicity. Proc. NatZ. Acad. Sci. USA 87: 4833-4835, 1990. GERBER, G., CERNE, R., AND RANDI~, M. Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in rat spinal dorsal horn. Brain Res. 56 1: 236-25 1, 199 1. GERBER, G., KANGRGA, I., RYU, P. D., LAREW, J. S. A., AND RANDI~, M. Multiple effects of phorbol esters in the rat spinal dorsal horn. J. Neurosci. 9: 3606-36 17, 1989. GERBER, G. AND RANDI~, M. Excitatory amino acid-mediated components of synaptically evoked input from dorsal roots to deep dorsal horn neurons in the rat spinal cord slice. Neurosci. Lett. 106: 2 1l-2 19, 1989. GREENAMYRE, J. T., YOUNG, A. B., AND PENNEY, J. B. Quantitative autoradiographic distribution of L[ 3H] glutamate-binding sites in rat central nervous system. J. Neurosci. 4: 2 133-2 144, 1984. Gu, Y. AND HUANG, L.-Y. M. Ca ions change the affinity of glycine to NMDA receptor complex. Sot. Neurosci. Abstr. 17: 1167, 199 1. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B., AND SIGWORTH, F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. PJliigers Arch. 39 1: 85-100, 1981. HARDY, J. D., WOLFF, H. G., AND GOODELL, H. Pain Sensations and Reactions. New York: Williams and Wilkins, 1952. Reprinted by Hafner, New York, 1967. HE~IMOVI~, H., RUSIN, K. I., RYU, P. D., AND RANDI~, M. Modulation of excitatory amino acid-induced currents by tachykinins and opioids in rat spinal cord neurons. Sot. Neurosci. Abstr. 16: 853, 1990. HONOR& T., DAVIES, S. N., DREJER, J., FLETCHER, E. J., JACOBSEN, P., LODGE, D., AND NIELSEN, F. E. Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science Wash. DC 241: 701-703, 1988. HUANG, L.-Y. M. Calcium channels in isolated rat dorsal horn neurones, including labelled spinothalamic and trigeminothalamic cells. J. Physiol. Lond. 411: 161-177, 1989. HUGANIR, R. L., DELCOUR, A. H., GREENGARD, P., HESS, G. P. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature Land. 32 1: 774-776, 1986. HUNT, S. P., PINI, A., AND EVAN, G. Induction of c-fos-like protein in CERNE,


MODULATION


spinal cord neurons following sensory stimulation. Nature Land. 328: 632-634, 1987. JOHNSON, J. W. AND ASCHER, P. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature Land. 325: 529-53 1, 1987. KANGRGA, I., GERBER, G., AND RANDI~, M. N-methyl-D-aspartate receptor-mediated component of fast and slow excitatory synaptic transmission in the rat spinal dorsal horn. Sot. Neurosci. Abstr. 14: 96, 1988. KANGRGA, I. AND RANDI~, M. Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice. J. Neurosci. 10: 2026-2038, 1990. KANGRGA, I. AND RANDI~, M. Outflow of endogenous aspartate and glutamate from the rat spinal dorsal horn in vitro by activation of low- and high-threshold primary afferent fibers. Modulation by p-opioids. Brain Res. 553: 347-352, 1991. KEMP, J. A., FOSTER, A. C., AND WONG, E. H. F. Non-competitive antagonists of excitatory amino acid receptors. Trends Neurosci. 10: 294-298, 1987. KLECKNER, N. W. AND DINGLEDINE, R. Requirement for glycine in activation of NMDA receptors expressed in Xenopus oocytes. Science Wash. DC241: 835-837, 1988. KUSHNER, L., LERMA, J., ZUKIN, R. S., AND BENNETT, M. V. L. Co-expression of N-methyl-D-aspartate and phencyclidine receptors in Xenopus oocytes injected with rat brain mRNA. Proc. Natl. Acad. Sci. USA 85: 3250-3254, 1988. LAMOTTE, C. C. AND DE LANEROLLE, N. C. Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. II. Methionine-enkephalin immunoreactivity. Brain Res. 274: 5 l-63, 1983. LEE, C.-M., CAMPBELL, N. J., WILLIAMS, B. J., AND IVERSEN, L. L. Multiple tachykinin binding sites in peripheral tissues and in brain. Eur. J. Pharmacol. 130: 209-2 17, 1986. LIPTON, S. A. AND KATER, S. B. Neurotransmitter regulation of neuronal outgrowth plasticity and survival. Trends Neurosci. 12: 265-270, 1989. LYNN, B. The heat sensitization of polymodal nociceptors in the rabbit and its independence of the local blood flow. J. Physiol. Land. 287: 493-507, 1979. MACDERMOTT, A. B., MAYER, M. L., WESTBROOK, G. L., SMITH, S. J., AND BARKER, J. L. NMDA-receptor activation increases cytoplasmatic calcium concentration in cultured spinal cord neurones. Nature Lond. 321: 519-522, 1986. MACDONALD, J. F., MODY, I., AND SALTER, M. W. Regulation of Nmethyl-D-aspartate receptors revealed by intracellular dialysis of murine neurones in culture. J. Physiol. Land. 414: 17-34, 1989. MALENKA, R. C., KAUER, J. A., PERKEL, D. J., AND NICOLL, R. A. The impact of postsynaptic calcium on synaptic transmission-its role in long-term potentiation. Trends Neurosci. 12: 444-450, 1989. MANTYH, P. W., PINNOCK, R. D., DOWNES, C. P., GOEDERT, M., AND HUNT, S. P. Correlation between inositol phospholipid hydrolysis and substance P receptors in rat CNS. Nature Lond. 309: 795-797, 1984. MAYER, E. A., Loo, D. D. F., SNAPE, W. J., JR., AND SACHS, G. The activation of calcium and calcium-activated potassium channels in mammalian colonic smooth muscle by substance P. J. Physiol. Land. 420: 47-71, 1990. MAYER, M. L., MACDERMOTT, A. B., WESTBROOK, G. L., SMITH, S. J., AND BARKER, J. L. Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using Arsenazo III. J. Neurosci. 7: 3230-3244, 1987. MAYER, M. L. AND MILLER, R. J. Excitatory amino acid receptors, second messengers and regulation of intracellular Ca2+ in mammalian neurons. In: The Pharmacology of Excitatory Amino Acids. A Special Report, edited by D. Lodge and G. Collingridge. Cambridge: Elsevier, 199 1, p. 36-42. MAYER, M. L., VYKLICK~, L., JR., AND CLEMENTS, J. Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine. Nature Lond. 338: 425-427, 1989. MAYER, M. L., WESTBROOK, G. L., AND GUTHRIE, P. B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature Land. 309: 261-263, 1984. MAYER, M. L. AND WESTBROOK, G. L. Permeation and block of Nmethyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones. J. Physiol. Lond. 394: 50 l-527, 1987a. MAYER, M. L. AND WESTBROOK, G. L. The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28: 197-276, 1987b. MEYER, R. A. AND CAMPBELL, J. N. Myelinated nociceptive afferents ac-

285

count for the hyperalgesia that follows a bum to the hand. Science Wash. 1981. R. L. Regulation of nicotinic acetylcholine receptors by protein phosphorylation. Mol. Neurobiol. 2: 9 l-l 24, 1988. MJELLEM-JOLY, N., LUND, A., BERGE, O.-G., AND HOLE, K. Potentiation of a behavioural response in mice by spinal co-administration of substance P and excitatory amino acid agonists. Neurosci. Lett. In press. MOCHLY-ROSEN, D., BASBAUM, A. I., AND KOSHLAND, D. E., JR. Distinct cellular and regional localization of immunoreactive protein kinase C in rat brain. Proc. Natl. Acad. Sci. USA 84: 4660-4664, 1987. MONAGHAN, D. T., BRIDGES, R. J., AND COTMAN, C. W. The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Annu. Rev. Pharmacol. Toxicol. 29: 365-402, 1989. MURASE, K. AND RANDI~, M. Electrophysiological properties or rat spinal dorsal horn neurones in vitro: calcium dependent action potentials. J. Physiol. Lond. 334: 141-153, 1983. MUFUSE, K. AND RANDI~, M. Actions of substance P on rat spinal dorsal horn neurones. J. Physiol. Lond. 346: 203-2 17, 1984. MURASE, K., RANDI~, M., AND RYU, P. D. Tachykinins modulate multiple ionic conductances in voltage-clamped rat spinal dorsal horn neurones. J. Neurophys. 61: 854-865, 1989a. MURASE, K., RAND& M., SHIRASAKI, T., NAKAGAWA, T., AND AKAIICE, N. Serotonin suppresses N-methyl-D-aspartate responses in acutely isolated spinal dorsal horn neurons of the rat. Brain Res. 525: 84-9 1, 1990. MURASE, K., RYU, P. D., AND RANDI~, M. Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci. Lett. 103: 56-63, 1989b. MURPHY, S. N., THAYER, S. A., AND MILLER, R. J. The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro. J. Neurosci. 7: 4145-4158, 1987. NICOLETTI, F., IADAROLA, M. J., WROBLEWSKI, J. T., ANDCOSTA, E. Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism. Developmental changes and interaction with cur-adrenoceptors. Proc. Natl. Acad. Sci. USA 83: 1931-1935, 1986a. NICOLETTI, F., MEEK, J. L., IADAROLA, M. J., CHUANG, D. M., ROTH, B. L., AND COSTA, E. Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus. J. Neurochem. 46: 40-46, 1986b. NISHIZUKA, Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature Lond. 308: 693-698, 1984. NISHIZUKA, Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature Lond. 334: 66 l-665, 1988. NOWAK, L. M., BREGESTOVS-k~, P., ASCHER, P., HERBET, A., AND PROCHIANTZ, A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature Lond. 307: 462-465, 1984. O’DONOHUE, T. L., HELKE, C. J., BURCHER, E., SHULTS, C. W., AND BUCK, S. H. Autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system. In: Substance Pand Neurokinins, edited by J. L. Henry, R. Couture, A. C. Cuello, G. Pelletier, R. Quirion, and D. Regoli. New York: Springer-Verlag, 1987, p. 93-95. O’MALLEY, P. J., CALLIGARO, D. O., AND MONN, J. A. Substance P and polyamines modulate the NMDA receptor operated ion channel by similar mechanisms. Sot. Neurosci. Abstr. 17: 1537, 199 1. PORTER, N. M., TWYMAN, R. E., UHLER, M. D., AND MACDONALD, R. L. Cyclic AMP-dependent protein kinase decreases GABA, receptor current in mouse spinal neurons. Neuron 5: 789-796, 1990. RANDI~, M., HE~IMOVI~, H., AND RYU, P. D. Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurons. Neurosci. Lett. 117: 74-80, 1990. RANDI~, M., MURASE, K., RYU, P. D., AND GERBER, G. Slow excitatory transmission in the rat spinal dorsal horn: possible mediation by tachykinins. Biomed. Res. 8, Suppl. : 7 l-82, 1987. REGOLI, D., DRAPEAU, G., DION, S., AND D’ORL~ANS-JUSTE, P. Pharmacological receptors for substance P and neurokinins. Life Sci. 40: 109117, 1987. RUSIN, K. I., BLEAKMAN, D., CHARD, P. S., MILLER, R. J., AND RANDI~, M. NMDA-evoked increases in [Ca2+]i in rat spinal cord neurons are enhanced by tachykinins. Sot. Neurosci. Abstr. 17: 983, 199 1. RUSIN, K. I., BLEAKMAN, D., CHARD, P. S., RANDI~, M., AND MILLER, R. J. Tachykinins potentiate N-methyl-D-aspartate responses in acutely isolated neurons from the dorsal horn. J. Neurochem. In press. RUSIN, K. I. AND RANDI~, M. Modulation of NMDA-induced currents DC213: 1527-1529, MILES, K. AND HUGANIR,


286


by p-opioid receptor agonist DAGO in acutely isolated rat spinal dorsal horn neurons. Neurosci. Lett. 124: 208-2 12, 199 la. RUSIN, K. I. AND RANDI~, M. Modulation of NMDA-induced currents by substance P: effects of spantide II and glycine. Sot. Neurosci. Abstr. 17: 983, 1991b. RYU, P. D., GERBER, J., MURASE, IS., AND RANDI~, M. Calcitonin generelated peptide enhances calcium current of rat dorsal root ganglion neurons and spinal excitatory synaptic transmission. Neurosci. Lett. 89: 305-312, 1988. RYU, P. D. AND RANDI~, M. Low- and high-voltage-activated calcium currents in rat spinal dorsal horn neurons. J. Neurophysiol. 63: 273285, 1990. SCHOEPP, D., BOCKAERT, J., AND SLADECZEK, F. Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors. In: The Pharmacology of Excitatory Amino Acids. A Special Report, edited by D. Lodge and G. Collingridge. Cambridge: Elsevier, 1991, p. 74-81. SKILLING, S. R., SMULLIN, D. H., BEITZ, A. J., AND LARSON, A. A. Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation. J. Neurochem. 51: 127-132, 1988. SLADECZEK, E., PIN, J. P., RECASENS, M., BOCKAERT, J., AND WEISS, S. Glutamate stimulates inositol phosphate formation in striatal neurones, Nature Lond. 317: 717-719, 1985. STRATTON, K. R., WORLEY, P. F., AND BARABAN, J. M. Excitation of hippocampal neurons by stimulation of glutamate Q, receptors. Eur. J. Pharmacol. 173: 235-237, 1989. STRATTON, K. R., WORLEY, P. F., AND BARABAN, J. M. Pharmacological characterization of phosphoinositide-linked glutamate receptor excitation of hippocampal neurons. Eur. J. Pharmacol. 186: 357-36 1, 1990. SUGIYAMA, H., ITO, I., AND HIRONO, C. A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature Lond. 325: 53 l533, 1987. SZEKELY, A. M., BARBACCIA, M. L., ALHO, H., AND COSTA, E. In primary cultures of cerebellar granule cells the activation of N-methyl-D-aspartate-sensitive glutamate receptors induces c-fos mRNA expression. Mol. Pharmacol. 35: 40 l-408, 1989. THOMSON, A. L. Glycine modulation of the NMDA receptor/channel complex. Trends Neurosci. 12: 349-353, 1989. TSIEN, R. Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures. Biochemistry 19: 2396-2404, 1980. URBAN, L. AND RANDI~, M. Slow excitatory transmission in rat dorsal horn: possible mediation by peptides. Brain Res. 290: 336-34 1, 1984. VYIUICK+, L., JR., BENVENISTE, M., AND MAYER, M. L. Modulation of

N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones. J. Physiol. Lond. 428: 3 13-33 1, 1990. WATKINS, J. C., KROGSGAARD-LARSEN, P., AND HONOR& T. Structureactivity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. In: The Pharmacology of Excitatory Amino Acids. A Special Report, edited by D. Lodge and G. Collingridge. Cambridge: Elsevier, 199 1, p. 4- 12. WATSON, S. P. AND DOWNES, C. P. Substance P induces hydrolysis of inositol phospholipids in guinea pig ileum and rat hypothalamus. Eur. J. Pharmacol. 93: 245-253, 1983. WIESENFELD-HALLIN, Z., H&FELT, T., LUNDBERG, J. M., FORSSMANN, W. G., REINECKE, M., TSCHOPP, F. A., AND FISCHER, J. Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses. Neurosci. Lett. 52: 199-204, 1985. WIESENFELD-HALLIN, Z., Xu, X.-J., AND DALSGAARD, C. J. Central sensitization after C-fiber activation is mediated by NMDA and substance P receptors. Neurosci. Abstr. 17: 728, 199 1. WOMACK, M. D., MACDERMOTT, A. B., AND JESSELL, T. M. Sensory transmitters regulate intracellular calcium in dorsal horn neurons. Nature Lond. 334: 351-353, 1988. WOMACK, M. D., MACDERMOTT, A. B., AND JESSEL, T. M. Substance P increases [ Ca]i in dorsal horn neurons via two distinct mechanisms. Sot. Neurosci. Abstr. 15: 184, 1989. WOOLF, C. J. AND THOMPSON, S. W. N. The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation: implications for the treatment of postinjury pain hypersensitivity states. Pain 44: 293-299, 199 1. WOOLF, C. AND WIESENFELD-HALLIN, Z. Substance P and calcitonin gene related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat. Neurosci. Lett. 66: 226-230, 1986. WORLEY, P. F., BARABAN, J. M., COLVIN, J. S., AND SNYDER, S. H. Inosito1 trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C. Nature Lond. 325: 159-16 1, 1987. YAKSH, T. L., JESSELL, T. M., GAMSE, R., MUDGE, A. W., AND LEEMAN, S. E. Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo. Nature Lond. 286: 155- 156, 1980. YOSHIMURA, M. AND JESSELL, T. M. Amino acid mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord. J. Physiol. Land. 430: 3 15-335, 1990. ZHENG, F. AND GALLAGHER, J. P. Trans-ACPD ( trans-D,L- 1-amino- 1,3cyclopentanedicarboxylic acid) elicited oscillation of membrane potentials in rat dorsolateral septal nucleus neurons recorded intracellularly in vitro. Neurosci. Lett. 125: 147-150, 199 1.


Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat.

Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid.

Lanthanum actions on excitatory amino acid-gated currents and voltage-gated calcium currents in rat dorsal horn neurons.

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in rat spinal dorsal horn.

Morphine withdrawal responses of rat locus coeruleus neurons are blocked by an excitatory amino-acid antagonist.

Excitatory amino acid modulation of lordosis in the rat.

Regional variation of excitatory and inhibitory amino acid-induced responses in rat dissociated CNS neurons.

Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

Uneven distribution of excitatory amino acid receptors on ventral horn neurones of newborn rat spinal cord.

Excitatory amino acid-mediated responses and synaptic potentials in medial pontine reticular formation neurons of the rat in vitro.

Developmental expression of excitatory amino acid responses in cerebellar Purkinje neurons in culture.

Excitatory amino acid immunoreactivity in vestibulo-ocular neurons in gerbils.

Differential modulation by dopamine of responses evoked by excitatory amino acids in human cortex.

Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat.

Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation.

Presynaptic inhibitory action of enkephalin on excitatory transmission in superficial dorsal horn of rat spinal cord.

Excitatory amino acid receptors appear to mediate paroxysmal depolarizing shifts in rat neocortical neurons in vitro.

Serotonin suppresses N-methyl-D-aspartate responses in acutely isolated spinal dorsal horn neurons of the rat.

Modulation of excitatory synaptic transmission in rat hippocampal CA3 neurons by triphenyltin, an environmental pollutant.

Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice.

Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated Ca1 neurons.

Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn.

Excitatory amino acid receptor activation produces a selective and long-lasting modulation of gene expression in hippocampal neurons.