Journal of Physiology (1995), 485.3, pp.787-796

Tachykininergic slow depolarization of motoneurones evokedby descending fibres in the neonatal rat spinal cord

Takashi Kurihara, Koichi Yoshioka * and Masanori Otsuka

Department of Pharmacology, Tokyo Medical and Dental University School of Medicine,1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan

1. In the isolated spinal cord of the neonatal rat, repetitive electrical stimulation of the uppercervical region elicited a prolonged depolarization of lumbar motoneurones (L3-5) lasting1-2 min, which was recorded extracellularly from ventral roots, or intracellularly.

2. This depolarizing response was markedly depressed by the excitatory amino acid receptorantagonists D-(-)-2-amino-5-phosphonovaleric acid (D-APV, 30 /M) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10/uM). The remaining response was further depressedby a 5-hydroxytryptamine (5-HT) receptor antagonist, ketanserin (3 /LM).

3. In the presence of these antagonists, a small part of the depolarizing response of slow timecourse remained, and this response was partially blocked by the tachykinin NK, receptorantagonists GR71251 (0 3-5 AM) and RP67580 (0 3-1 /SM). In contrast, RP68651(0 3-1 /sM), the inactive enantiomer of RP67580, had no effect on the depolarizing response.

4. The slow depolarizing response in the presence of D-APV, CNQX and ketanserin wasmarkedly potentiated by a peptidase inhibitor, thiorphan (1 /SM).

5. This descending fibre-evoked slow depolarization became smaller after prolonged treatment(5-7 h) with 5,7-dihydroxytryptamine (10 #M), a neurotoxin for 5-HT neurones. Undersuch conditions, the effects of thiorphan and GR71251 on the slow depolarization werevirtually absent.

6. Under the action of D-APV, CNQX and ketanserin, applications of tachykinins, substanceP and neurokinin A produced depolarizing responses of lumbar motoneurones, and theresponses were depressed by GR71251 and potentiated by thiorphan.

7. These results suggest that tachykinins contained in serotonergic fibres serve as neuro-transmitters mediating the descending fibre-evoked slow excitatory postsynaptic potentialsin motoneurones.

Two mammalian tachykinins, substance P (SP) andneurokinin A (NKA), are present in certain populations ofcentral and peripheral neurones and may serve as neuro-transmitters (for review, see Otsuka & Yoshioka, 1993).There is good evidence for the neurotransmitter role oftachykinins in sympathetic ganglia (Konishi, Tsunoo &Otsuka, 1979; Tsunoo, Konishi & Otsuka, 1982) andenteric plexus (Johnson, Katayama, Morita & North, 1981).In guinea-pig prevertebral ganglia the tachykinins releasedfrom collaterals of certain primary afferent fibres contributeto the generation of slow excitatory postsynaptic potentials(EPSPs). On the other hand, the role of tachykinins asneurotransmitters in the central nervous system is lessclear. In the mammalian spinal cord, the tachykinin-containing primary afferent fibres mainly terminate in the

dorsal horn and it is conceivable that they also evoke similarslow EPSPs in second-order neurones. Studies with youngrat spinal cord slices by Randic and colleagues showed thatSP- and dorsal root-evoked slow depolarizations of dorsalhorn cells recorded intracellularly were blocked bytachykinin antagonists or SP antibodies (Urban & Randic,1984; Randic, Ryu & Urban, 1986). De Koninck & Henry(1991) showed that slow EPSPs recorded from cat dorsalhorn neurones in vivo following noxious cutaneousstimulation were blocked by intravenous injection of atachykinin antagonist, CP-96,345. These and many otherstudies suggest that tachykinins, released from primaryC-afferent terminals, produce slow EPSPs in second-orderneurones in the spinal dorsal horn. However, the directdemonstration of tachykininergic slow EPSPs in the spinal

* To whom correspondence should be addressed.

3537 787

7T Kurihara, K. Yoshioka and M. Otsuka

dorsal horn appears to be still incomplete, because the slowEPSPs recorded from dorsal horn neurones in these studiesmay be produced by polysynaptic pathways includingtachykininergic neurones.

SP is also present in the ventral horn in considerableamounts, although its concentration is much less than thatin the superficial dorsal horn (Jessell, Tsunoo, Kanazawa &Otsuka, 1979; Kanazawa, Sutoo, Oshima & Saito, 1979).The content of SP in the ventral horn was markedlyreduced by spinal transection at upper levels, whereas itwas only slightly reduced by dorsal rhizotomy (Kanazawaet al. 1979). The serotonergic descending fibres whichoriginate from the caudal ventral medulla have been shownto contain SP (Chan-Palay, Jonsson & Palay, 1978; H6kfelt,Ljungdahl, Terenius, Elde & Nilsson, 1978) and NKA(Franck, Brodin, Fried, Rosen, Yamamoto & Fried, 1991).Immunocytochemical studies have demonstrated thatsome of the SP-containing fibres form synapses withmotoneurones (Pelletier, Steinbusch & Verhofstad, 1981;De Lanerolle & LaMotte, 1982; Vacca, Hobbs, Abrahams &Naftchi, 1982; Ulfhake et al. 1987).

The isolated spinal cord preparations of neonatal ratsenable us to obtain stable extracellular and intracellularrecordings from motoneurones and thus to carry outdetailed pharmacological analyses of both the responses tonerve stimulation and responses to exogenously appliedagents. In this preparation our group and other investigatorsrecorded extracellularly or intracellularly a depolarizingresponse of slow time course from motoneurones evoked byelectrical stimulation of upper parts of the spinal cord(Takahashi, 1985; Yomono, Suzuki & Yoshioka, 1992;Elliott & Wallis, 1993; Wallis & Wu, 1993). In the presentstudy we examined whether tachykinins contribute to thedescending fibre-evoked slow depolarization of lumbarmotoneurones in the neonatal rat spinal cord.

A preliminary report of this study has appeared as anabstract (Kurihara, Yoshioka & Otsuka, 1993).

METHODSPreparationThe isolated spinal cord preparation of the neonatal rat was usedas described previously (Yomono et al. 1992). Under etheranaesthesia, the spinal cord below upper cervical level (C1-4) wasremoved together with the vertebral column from 0- to 6-day-oldWistar rats of either sex and placed in a dissecting dish filled withartificial cerebrospinal fluid (ACSF) saturated with 95% 02 and5% Co2. The composition of ACSF was as follows (mM): 138 6NaCl, 3-35 KCl, 1-25 CaC12, 1P15 MgCl2, 20-9 NaHCO3 and 10 0glucose. Ventral laminectomy was performed and the spinal cord,with attached L3-5 ventral and dorsal roots, was isolated. Thecord below upper cervical level was placed in a recording chamberof 0-6 ml volume coated with Sylgard (Dow Corning) and perfused(2-5 ml min-) with ACSF saturated with 95% 02 and 5% CO2 atroom temperature (24-25 0C). For intracellular recordings, the

spinal cord was hemisected and the pial membrane on the lateralsurface of the lumbar region was removed with fine scissors tofacilitate penetration with microelectrodes. With the lateral sideupward, the hemisected cord was immobilized with small pins onthe bottom of the chamber.

In the experiments to construct concentration-response curves toSP, NKA and thyrotropin-releasing hormone (TRH), hemisectedspinal cords below middle thoracic level were isolated by a similarmethod and placed in a smaller recording chamber of 0 3 mlvolume to facilitate exchange of perfusion medium.

Extracellular recordingsElectrical stimuli (usually with standard parameters: twentypulses of 60 V in amplitude and 2 ms in duration at 10 or 50 Hz;see Results) were applied to the upper cervical region (C1-4) every15 min and the resulting potential changes were recordedextracellularly from a ventral root (L3-5). Simultaneous recordingsfrom a ventral root extracellularly, and a motoneurone of the samesegment intracellularly, have demonstrated that the potentialchanges at both locations had a very similar pattern (Konishi &Song, 1987), which suggests that the ventral root recording reflectsthe electrical activities of motoneurones. Potential changes of theventral root were led to a DC amplifier and then to a chart recorderand a computer recording device (Axotape version 2, AxonInstruments). Stimulation and recording were carried out withtightly fitting suction electrodes. The magnitude of thedepolarizing response to the electrical stimulation was expressedas the integrated area of the depolarization. Involvement oftachykinins in the depolarizing response induced by descendingfibre stimulation was examined under the action of the excitatoryamino acid (EAA) receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (D-APV) at 30,uM and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at 10 FM, and the5-hydroxytryptamine (5-HT) receptor antagonist ketanserin at3 FM. The effects of EAA receptor antagonists and ketanserinwere evaluated by comparing the averaged magnitude of threesuccessive responses before and after equilibration with the drugs.A 10 min exposure to the EAA receptor antagonists of the spinalcord was sufficient to obtain a steady depressant effect, whereasmore than 30 min exposure to ketanserin was necessary for its fulleffect.

In order to examine the effects of the tachykinin receptorantagonist, GR71251, on the depolarizing responses to SP, NKAand TRH of motoneurones, these agonists were bath applied tothe spinal cord for 30 s at increasing concentrations with intervalsof 15-20 min (SP and NKA) or 25 min (TRH) to avoidtachyphylaxis in the presence of D-APV (30 FM), CNQX (10 FM)and ketanserin (3 FM). After constructing the controlconcentration-response curve, the spinal cord was allowed toequilibrate for 10 min with GR71251 (0-3 FM against NKA,3 FM against SP, and 5 FM against TRH) and theconcentration-response curve to each peptide agonist in thepresence of GR71251 was again constructed. The magnitude ofeach response was measured as the integrated area ofdepolarization.

In the experiments to examine the effects of a peptidase inhibitor,thiorphan (1 FM), on the depolarizations induced by descendingfibre stimulation and peptide agonists, a stock solution ofthiorphan (5 mM) was diluted to the final concentration with ACSFimmediately before use to avoid the loss of action.

788 J Physiol.485.3

Tachykininergic slow EPSPs

Intracellular recordingsIntracellular recordings from lumbar motoneurones (L3-5) weremade with glass microelectrodes filled with 3 M potassium acetate.The DC resistance of the electrodes was between 60 and 100 M.Q.Impalement was usually facilitated by over-compensating thecapacitance neutralization circuit of a microelectrode amplifier(MEZ 8201, Nihon Kohden, Tokyo, Japan). Motoneurones wereidentified by antidromic action potentials evoked by ventral rootstimulation with suction electrodes using square pulses of 1-30 Vamplitude and 300-500 jus duration. The resting membranepotential was continuously monitored on the chart recorder andthe computer recording device. Electrical stimulation withstandard parameters (see above) was applied to the upper cervicalregion every 8 min and the resulting potential changes wererecorded. Contribution of tachykinins to the depolarization evokedby the descending fibre stimulation was examined in the same wayas in the experiments with extracellular recordings.

DrugsThe following drugs were used: CNQX and ketanserin tartrate werepurchased from Research Biochemicals Inc., Natick, MA, USA;D-APV was from Cambridge Research Biochemicals Ltd, Cheshire,UK; 5,7-dihydroxytryptamine creatinine sulphate, DL-thiorphanand naloxone hydrochloride were from Sigma; NKA, SP and TRHwere from Peptide Institute Inc., Osaka, Japan; GR71251,[D-Pro9[spiro-y-lactam]Leu10,Trp11]SP, was kindly donated byDr R. M. Hagan, Department of Neuropharmacology, Glaxo GroupResearch Ltd, Herts, UK; RP67580, (3aR,7aR)-7,7-diphenyl-2- [1-imino-2-(2-methoxyphenyl)-ethyl]perhydroisoindol-4-one andRP68651, (3aS,7aS-7,7-diphenyl-2-[1-imino-2-(2-methoxyphenyl)-ethyl]perhydroisoindol-4-one, were kindly donated by Dr C. Garret,Rh8ne-Poulenc Rorer, Centre de Recherches de Vitry-Alfortville,Vitry-sur-Seine, France. All drugs were dissolved in ACSF andapplied to spinal cords by perfusion.

Statistical analysisStatistical analysis was performed using Student's t test.A P value less than 0 05 was considered significant.

AControl

BD-APVCNQX

RESULTSDescending fibre-evoked slow depolarization ofventral roots in normal ACSF and in the presence ofexcitatory amino acid receptor antagonists andketanserinWhen the spinal cord was perfused with normal ACSF, asingle shock of 60 V in amplitude and 2 ms in duration(supramaximum intensity) given to the upper cervicalregion evoked a fast depolarizing response of lumbarventral roots of approximately 2 mV in amplitude, followedby a smaller and much slower depolarization. Whenrepetitive electrical stimulation (twenty pulses of 60 V and2 ms at 10 or 50 Hz) was applied, the latter slower responsebecame more pronounced and lasted about 1-2 min(Fig. 1A). This response will be referred to as thedescending fibre-evoked slow ventral root potential (VRP).

The descending fibre-evoked slow VRP presumablyrepresents EPSPs in motoneurones evoked by bothmonosynaptic inputs from descending fibres and trans-synaptic inputs. In order to suppress the componentsmediated by excitatory amino acid (EAA) receptors in bothmonosynaptic and trans-synaptic inputs (Kurihara,Yoshioka & Otsuka, 1991; Elliott & Wallis, 1993; Wallis &Wu, 1993), the preparation was treated with EAA receptorantagonists (a mixture of 30 /SM D-APV and 10 /SM CNQX).The descending fibre-evoked slow VRP was markedlydepressed by the mixture of EAA receptor antagonists toabout 40% of the control response (Fig. 1B). Previously,our group and other investigators observed thatdescending fibre-evoked slow VRP was sensitive to the5-hydroxytryptamine (5-HT) receptor antagonistketanserin (Yomono et at. 1992; Wallis & Wu, 1993). In thepresence of the EAA receptor antagonists, ketanserin(3 /SM) further decreased the remaining response, so that

C+ Ketanserin

I3 mV

1 min

Figure 1. Descending fibre-evoked slow ventral root potential in normal medium and in thepresence of excitatory amino acid receptor antagonists and ketanserinElectrical stimulation with twenty pulses of 60 V and 2 ms at 10 Hz was applied to the upper cervicalregion every 15 min, and the resulting potential changes were recorded extracellularly from a L4 ventralroot. A, control response; B, response in the presence of EAA receptor antagonists D-APV (30 /M) andCNQX (10 /M); C, response after adding ketanserin (3/uM) in the continued presence of the EAA receptorantagonists.

J Phy8iol. 485.3 789

I

i

T Kurihara, K. Yoshioka and M. Otsuka

the mixture of D-APV (30 /uM), CNQX (10 ,UM) andketanserin (3 /SM), reduced the descending fibre-evokedslow VRP to 18-5 + 1P49% of the control response(mean+s.E.M., n=23;Fig.lC).

The remaining depolarizing response in the presence ofD-APV, CNQX and ketanserin had a slow time course(1-2 min) and the size of the response became larger withincreases in the frequency (up to 50 Hz), number of pulses(up to thirty pulses) and pulse width (up to 2 ms). In theexperiments described below we used the stimulationparameters of twenty pulses of 2 ms duration at 10 or 50 Hzwith 15 min intervals (except for intracellular recordings;see Methods), which gave stable responses for at least 4 h.

A

Effects of tachykinin antagonists on the descendingfibre-evoked slow depolarizationTo test whether tachykinins contribute to the descendingfibre-evoked slow VRP that remained under the action ofEAA and 5-HT receptor antagonists, we examined theeffects of a peptide tachykinin NK1 receptor antagonist,GR71251 (Ward, Ewan, Jordan, Ireland, Hagan & Brown,1990; Guo, Yoshioka, Yanagisawa, Hosoki, Hagan &Otsuka, 1993) and a non-peptide tachykinin NK1 receptorantagonist, RP67580 (Garret et al. 1991; Hosoki,Yanagisawa, Guo, Yoshioka, Maehara & Otsuka, 1994). Inthe presence of the mixture of D-APV (30 ,uM), CNQX(10 /SM) and ketanserin (3 UM), GR71251 (0 3-5 #M)

B

a

mV

1 min

110 r

100 I

90 k

t*r t80 F

tt

t

70 F

60 F

50 F1 0-3 5 11 3

GR71 251

40L0 50 100

Time (min)

I 5150 200

Figure 2. Effect of GR71251 on the descending fibre-evoked slow ventral root potential (VRP)A, sample records from a single experiment. A a, control response; A b, 8 min after adding GR71251(3/uM); A c, 37 min after washing out GR71251. The stimulation with twenty pulses of 60 V and 2 ms at50 Hz was applied to the upper cervical region every 15 min and the resulting potential changes were

recorded from a L4 ventral root. B, time course of the depressant effect of GR71251 at increasingconcentrations on the descending fibre-evoked slow VRP. GR71251 (0 3-5 AM) was cumulatively applied.Ordinate: the magnitude of descending fibre-evoked slow VRP was measured as the integrated area ofdepolarization and expressed as percentage of the averaged magnitude of the first three consecutivecontrol responses immediately before application of GR71251. The conditions of stimulation andrecording were the same as in Fig. 1. Each point and vertical bar represent the mean + S.E.M. (n = 6).*P < 005, t P < 001, when compared with the control value. Ketanserin (3 FM), D-APV (30 FM) andCNQX (10 FM) were present in the perfusion medium throughout the experiments.

GR71 251 1-1

0c

Coa.a)

3:0CuCu

c Wash

J Physiol.485.3790

Tachykininergic

significantly depressed the descending fibre-evoked slowVRP (Fig.2). When the concentration of GR71251 wascumulatively increased, its depressant effect was evident at0-3 /SM, and at 5 uM the antagonist depressed the slowVRP to 587 + 3-6% of the control response (n= 6,P < 0-01). RP67580 (0 3-1 ,UM) likewise significantlydepressed the slow VRP to 58-8 + 2 8% at 1 /,M (n = 5,P < 0 001). In contrast, RP68651 (0 3-1 juM), the inactiveenantiomer of RP67580 (Garret et al. 1991), had no effecton the slow VRP (100-4 + 2-6% of the control response at1/uM; n=5).

Similar results were obtained with intracellular recordingsfrom lumbar motoneurones. In normal medium, electricalrepetitive stimulation of descending fibres evoked fastdepolarizing responses usually accompanied by spikedischarges, followed by a long-lasting depolarization. In thepresence of EAA receptor antagonists and ketanserin, thedepolarization became much smaller, with a peak amplitude

slow EPSPs 791

of 2-5 mV. The effect of GR71251 on the remaining slowdepolarization, which probably represents an EPSP, wasexamined in ten motoneurones that had stable restingmembrane potentials between -65 and -80 mV. Figure 3illustrates typical results. GR71251 (3,UM) depressed thedescending fibre-evoked slow EPSP to 49 0 + 7 5%(n = 10) of the control in a reversible manner (Fig. 3A b andB). In particular, the later part of the response wasmarkedly depressed by GR71251 (Fig. 3A d). GR71251 hadno effect on the resting membrane potential or inputresistance of motoneurones.

Effects of thiorphan on the descending fibre-evokedslow depolarizationPrevious studies in our laboratory showed that peptidaseinhibitors potentiated the tachykininergic components ofprimary afferent-evoked ventral root responses in theneonatal rat spinal cord, which suggested that enzymaticdegradation plays a role in the termination of transmitter

BA

120 F

bGR71 251

c dWash a + b

f r _ f rn1 min

0~

a-w

0

60(a02

Figure 3. Intracellular recordings showing the effect of GR71251 on the descending fibre-evoked slow EPSPs recorded from motoneuronesA, sample records from a single experiment. A a, control response; A b, 12 min after adding GR71251(3/uM); Ac, 20 min after washing out GR71251; Ad, superimposition of records in A a and A b. B, timecourse of the depressant effect of GR71251 on the descending fibre-evoked slow EPSPs in motoneurones.GR71251 (3/SM) was applied during the period indicated by the horizontal bar. Ordinate: the magnitudeof descending fibre-evoked slow EPSP was measured as the integrated area of depolarization andexpressed as percentage of the averaged magnitude of the three consecutive control responses

immediately before application of GR71251. Ketanserin (3 /M), D-APV (30 /M) and CNQX (10 /SM) were

present in the perfusion medium throughout the experiments. The stimulation with twenty pulses of 60 Vand 2 ms at 10 Hz was applied to the upper cervical region every 8 min. Resting membrane potential was-85 to -80 mV. Each point and vertical bar represent the mean ± s.x.M. (n = 10). *P< 0 05, t P< 0 01,t P < 0-001, when compared with the control value.

J Physiol.485.3

aControl

100 I

80 F *

60 F

40 -

GR71 251

20 L.

0 10 20 30 40 50

Time (min)60 70 80

792 T Kurihara, K. Y(

action of tachykinins in this preparation (Suzuki et al.1994). We therefore examined the effects on the descendingfibre-evoked slow VRP of thiorphan (1 /M), a specificinhibitor of enkephalinase (endopeptidase-24.1 1) (Roques etal. 1980). The experiments were carried out in the presenceof naloxone (1 /SM), because the presence of opioid peptidesin the serotonergic descending fibres has been reported(Millhorn, HMkfelt, Verhofstad & Terenius, 1989), and theblockade of enzymatic degradation of tachykinins and thatof opioid peptides by thiorphan might result in oppositeeffects cancelling each other.

The descending fibre-evoked slow VRP was markedlypotentiated and prolonged by thiorphan (Fig. 4A a). Themagnitude of the slow VRP under thiorphan (1 uM) was

A

oshjioka and M. Otsuka J Physiol.485.3

about 155% of the control (Fig. 4B; n = 4). In the presenceof thiorphan, GR71251 (3 /tM) depressed the slow VRP toabout 50% of the response immediately before applicationof the antagonist (Fig. 4A b and B). Intracellular recordingsalso confirmed the potentiating action of thiorphan on thedescending fibre-evoked slow EPSP and the depressantaction of GR71251 upon it (n = 2, not shown).

Effects of 5,7-dihydroxytryptamine on thepotentiating action of thiorphan and the depressantaction of GR71251To examine whether the tachykinins involved in thedescending fibre-evoked slow VRP are those contained inserotonergic descending fibres, we examined the effects of5,7-dihydroxytryptamine (5,7-DHT), which is a toxin for

B

Control

b

Naloxone

* Thiorphan

* t GR71251

0 50 100 150Time (min)

Figure 4. Effects of thiorphan and GR71251 on the descending fibre-evoked slow ventral rootpotential (VRP) in preparations with or without pretreatment with 5,7-dihydroxytryptamine(5,7-DHT)A, sample records from a single experiment in a preparation without pretreatment of 5,7-DHT.A a, superimposed records of control response and the response after adding thiorphan (1 /M);A b, superimposed records of the response under the action of thiorphan (same as shown A a) and thatafter further addition of GR71251 (3 /M). B, time course of the effects of naloxone, thiorphan andGR71251 on descending fibre-evoked slow VRP in untreated preparations (0) and those treated with5,7-DHT (0). Experiments were carried out on four untreated preparations and three preparations whichhad been pre-incubated with 5,7-DHT (10 ,uM). Naloxone (1 uM), thiorphan (1 M) and GR71251 (3/uM)were applied during the periods indicated by the horizontal bars. Ordinate: the magnitude of descendingfibre-evoked slow VRP was measured as the integrated area of depolarization and expressed as percentageof the averaged magnitude of three consecutive responses immediately before application of naloxone.Ketanserin (3 AM), D-APV (30 FM) and CNQX (10 /SM) were present in the perfusion medium throughoutthe experiments. The conditions of stimulation and recording were the same as in Fig. 1. Each point andvertical bar represent the mean + S.E.M. Vertical bars are only indicated when larger than symbols.*P < 0 05, t P < 0-01, I P < 0-001, when compared with the control response.

180 r

160 p

140 F

Thiorphan

00-

Cuccco.I-

coa)D-

120 k

100 -1 mv

1 min

GR71 251

80 F

60 F

t

200 250

Ca

// Thio,phan

No m-

a

Tachykininergi(

5-HT neurones (Baumgarten, Klemm, Lachenmayer,Bjorklund, Lovenberg & Schlossberg, 1978). The isolatedspinal cord was continuously perfused with ACSFcontaining 10 /M 5,7-DHT for 5 h. The mixture of D-APV,CNQX and ketanserin was then added to perfusionmedium. Yomono et al. (1992) found that continuousapplication of 10 #M 5,7-DHT to the spinal cord for 4-6 hresulted in a marked decrease in the 5-HT content, to 30%of the control level, with little effect on the norepinephrinecontent and at the same time in a blockade of the long-lasting descending inhibition of the monosynaptic reflex.

Aa b

c slow EPSPs 793

After treatment with 5,7-DHT, the descending fibre-evoked slow VRP became smaller than that observed in thepreparations that had not been treated with 5,7-DHT. Theaverage magnitude of the slow VRP in the treatedpreparation was 0-132 + 0 005 mV min (n = 4), whereasthat in the untreated preparation was 0-381 + 0-021mV min (n = 14; P< 0 001). Furthermore, the potentiatingeffect of thiorphan (1 /zM) and the depressant effectGR71251 (3/uM) on the descending fibre-evoked slow VRPwere virtually absent (Fig. 4B).

c

5 r

4

3

2

0-03 0.1 0-3 1.0

[SP] CUM)

o L0-03

5-

4-

3-

t 2 -

0-1 0-3 1.0 0-03

[NKA] (juM)0-1 0-3 1.0

[TRH] (uM)

a

-[i~~~~~~~==~~

b

120

100

80

60

40

20

00-03 0-1 0-3 1-0 0-03

Concentration (uM)0-1

Concentration (uM)0-3

Figure 5. Effects of GR71251 and thiorphan on the concentration-response curves to SP, NKAand TRHA, effects of GR71251 on SP- (A a), NKA- (A b) and TRH-evoked (Ac) responses. The area ofdepolarization in mV min was plotted against logarithmic concentrations of the peptides. Open symbolsindicate the control responses and filled symbols those after addition of GR71251 at 3/uM in A a, at0-3 /SM in A b, and at 5 /SM in A c. *P < 0 05, t P < 0 01, when compared with the control response evokedby an agonist at the corresponding concentration. B, effects of thiorphan on SP- (O and 0), NKA-(n and *), and TRH-evoked responses (A and A). Open symbols indicate the magnitude of depolarizationbefore addition of thiorphan and filled symbols those 10 min after addition of thiorphan (1 /uM). Ordinate:area of depolarization expressed as percentage of the area of control response in mV min evoked by 1 /,MSP, 1 /LM NKA and 0 3 /SM TRH. Abscissa: logarithmic concentration of SP, NKA and TRH. *P < 0 05,t P < 0-01, I P < 0.001, when the ratio of the magnitude of NKA-evoked responses after addition ofthiorphan to that before thiorphan was compared with the corresponding ratio for SP-evoked responses ateach agonist concentration. Concentration-response curves were first constructed in the presence ofketanserin (3 /ZM), D-APV (30 /M) and CNQX (10 /sM), and then after further additions of GR71251 or

thiorphan in the same preparations. The peptide agonists were applied by perfusion for 30 s. Each pointand vertical bar represent the mean + S.E.M. (n = 3). Vertical bars are indicated only when larger thansymbols. The positions of some symbols are horizontally adjusted to avoid overlapping.

J Physiol. 485.3

:5

4

3

2

0

0~coN

.-

c_$._

aa)

B

c0co

coN

Cu._

a) 100

0

500r

400

300

200

4T Kurihara, K. Yoshioka and M. Otsuka

Effects of GR71251 on SP-, NKA- and TRH-inducedventral root depolarizationsIn the presence of D-APV, CNQX and ketanserin, bathapplications of SP, NKA and TRH to the spinal cordproduced concentration-dependent depolarizations of lumbarventral roots (Fig. 5A). The responses to SP (Fig. 5A a) andNKA (Fig. 5A b) were depressed by GR71251 (3/Magainst SP and 0 3 uM against NKA, respectively). Theantagonistic action of GR71251 against SP wasapproximately one order of magnitude weaker than thatagainst NKA. Thus 0 3 /LM GR71251 was sufficient to blockalmost completely the action of NKA at up to 041 /M,whereas a tenfold higher concentration of the antagonistwas necessary to obtain a comparable blockade of theaction of SP. In contrast, the TRH-induced depolarizationwas not depressed, but slightly potentiated by GR71251(3-5/uM; Fig. 5A c).

Effects of thiorphan on SP-, NKA- and TRH-induced depolarizationsThe depolarizing responses of lumbar ventral roots to bathapplications of SP and NKA were markedly augmented by1 /SM thiorphan (Fig. 5Ba). In contrast, thiorphan did notpotentiate the depolarizing action of TRH (Fig. 5B b). Theresponses to NKA were more markedly potentiated bythiorphan than those to SP

DISCUSSIONThe present study suggests that tachykinins contained inserotonergic descending fibres produce slow EPSPs inmotoneurones in the neonatal rat spinal cord. Thedescending fibre-evoked slow depolarization of motoneuronesobserved in the presence of the EAA receptor antagonistsand ketanserin was partially depressed by GR71251 orRP67580, and potentiated by thiorphan. This componentof the slow depolarization, which is sensitive to tachykininreceptor antagonists and thiorphan, is likely to be due to adirect action of tachykinins on motoneurones, because (1)polysynaptic components are probably blocked by the EAAantagonists, and (2) there is morphological evidence thatSP-containing axonal boutons form synapses withdendrites of motoneurones (see introduction).

In this study, electrical stimulation was applied to theupper cervical region of the spinal cord. This maystimulate, in addition to bulbospinal descending fibres,other fibre pathways as well. Since primary afferentstimulation evokes slow depolarizing responses of similartime course in motoneurones (Akagi, Konishi, Otsuka &Yanagisawa, 1985; Nussbaumer, Yanagisawa & Otsuka,1989), the slow depolarization observed in the presentstudy might be due to stimulation of primary afferent fibresin the cervical segments and subsequent activation of spinalinterneurones. This possibility appears unlikely, however,

because the slow depolarization evoked by primary afferentstimulation was almost completely blocked by the mixtureof EAA receptor antagonists used in this study (Kuriharaet al. 1991). Furthermore, we observed that the 5,7-DHTtreatment depressed the descending fibre-evoked slow VRPand virtually abolished the potentiating action of thiorphanand the depressant action of GR71251 on the slow VRP.Therefore it is likely that the tachykinins responsible forthe slow EPSP are mainly contained in serotonergicdescending fibres.

Guo et al. (1993) and Hosoki et al. (1994) examined thepharmacological profiles of GR71251 and RP67580 in theneonatal rat spinal cord and showed that they were specifictachykinin NK1 receptor antagonists. Furthermore,RP68651, the inactive enantiomer of RP67580, had noeffect on the descending fibre-evoked slow VRP. Thedepressant effects of GR71251 and RP67580 on thedescending fibre-evoked slow depolarization of motoneuronesare therefore probably due to their blocking actions ontachykinin NK1 receptors. The contributions of NK2 andNK3 tachykinin receptors to the tachykininergic slowEPSP may be negligible, because our previous studies haveshown that both NK2 and NK3 agonists had little or nodepolarizing actions on neonatal rat spinal motoneurones(Yanagisawa & Otsuka, 1990; Guo et at. 1993). Thesestudies, however, also suggested the possible existence ofNK1 receptor subtypes on motoneurones of the neonatalrat spinal cord (Yanagisawa & Otsuka, 1990; Guo et al.1993; Hosoki et at. 1994). Therefore further studies arenecessary to clarify the pharmacological characteristics ofthe tachykinin receptors responsible for the descendingfibre-evoked slow depolarization.

In the experiment in which the effect of cumulativeapplication of GR71251 on the descending fibre-evokedslow VRP was examined, we noticed that the slow VRPtended to be depressed biphasically by low (0'3-1 /M) andhigh (3-5 /M) concentrations of GR71251 (Fig. 2B). It istempting to suppose that the depressant effects ofGR71251 at the low and high concentrations are mainlydue to its antagonism against NKA and SP, respectively,since the potencies of SP and NKA to evoke depolarizationof motoneurones were approximately equal, and GR71251antagonized the action ofNKA more potently than that ofSP in the presence of the EAA receptor antagonists andketanserin (see Fig. 5A). This hypothesis, however, needsfurther verification.

After the treatment with EAA receptor antagonists andketanserin, the descending fibre-evoked slow depolarizationwas depressed by either GR71251 or RP67580 to only50-60% of the control. One of the candidates for neuro-transmitters which may contribute to the remaining slowdepolarization is TRH, since extensive co-existence of SP,

794 J Physiol.485.3

J Physiol. 485.3 Tachyklininergic slow EPSPs 795

5-HT and TRH was demonstrated in the descending fibresfrom caudal raphe nuclei (Johansson et al. 1981), andTakahashi (1985) demonstrated that an antisera againstTRH depressed the slow depolarization of lumbarmotoneurones evoked by the descending pathway in thesame preparation. It is also conceivable that the serotonergiccomponent was not completely blocked by ketanserin, aswe observed that ketanserin did not completely abolish thedepolarization of motoneurones evoked by exogenouslyapplied 5-HT in this preparation (K. Yoshioka & T.Kurihara, unpublished observation). The contribution ofcatecholamines (norepinephrine, epinephrine and dopamine)appears small, because the a-adrenergic receptorantagonist, phentolamine (10-30 /LM), did not block theslow depolarization under the action of EAA receptorantagonists and ketanserin (T. Kurihara & K. Yoshioka,unpublished observation; see also Wallis & Wu, 1993).Among neuropeptides, vasopressin, in addition to TRHand tachykinins, has a direct depolarizing action on theneonatal rat spinal motoneurones (Suzue, Yanaihara &Otsuka, 1981). However, it was shown that vasopressin-containing fibres, which are derived predominantly fromthe paraventricular nucleus, innervate mainly thesubstantia geratinosa and the intermediolateral column(Buijis, 1978).

Thiorphan markedly potentiated the descending fibre-evoked slow depolarizing response as well as thedepolarizing actions of exogenous SP and NKA. Sincethiorphan is known to be an inhibitor of enkephalinase(Roques et al. 1980), these results suggest that this enzymeis involved in terminating the transmitter action oftachykinins released from descending fibres. Althoughimmunohistochemical studies have shown the co-existenceof enkephalins with SP and TRH in the serotonergicdescending fibres in the rat (Millhorn et al. 1989), we didnot observe any noticeable effect of naloxone (1-5 uM) onthe descending fibre-evoked slow VRP (Fig. 4B).

The functional significance of the slow depolarizationevoked by tachykinins (SP and NKA) presumablyco-released with 5-HT and TRH remains to be clarified (forreview, see Holstege & Kuypers, 1987; Jacobs & Azmitia,1992). All of these neurotransmitters have depolarizingactions on motoneurones. Furthermore, SP and TRH wereshown to have potentiating effects on the excitatory actionof 5-HT on the motoneurone membrane. Presynapticinteractions between 5-HT and SP have also been suggestedin the rat spinal cord ventral horn. It has been suggestedthat 5-HT-containing descending fibres from caudal raphenuclei exert 'gainsetting' action, enhancing the overallresponsiveness of motoneurones (Holstege & Kuypers, 1987).Further studies are needed to understand differentialfunctional roles of tachykinins and other co-transmittersreleased from these bulbospinal fibres.

AKAGI, H., KoNISHI, S., OTSUKA, M. & YANAGISAWA, M. (1985). Therole of substance P as a neurotransmitter in the reflexes of slow timecourse in the neonatal rat spinal cord. British Journal ofPharmacology 84, 663-673.

BAUMGARTEN, H. G., KLEMM, H. P., LACHENMAYER, L., BJORKLUND,A., LOVENBERG, W. & SCHLOSSBERG, H. G. (1978). Mode andmechanism of action of neurotoxic indoleamines: A review and aprogress report. Annals of the New York Academy of Sciences 305,3-24.

Buijis, R. M. (1978). Intra- and extrahypothalamic vasopressin andoxytocin pathways in the rat. Pathways to the limbic system,medulla oblongata and spinal cord. Cell and Tissue Research 192,423-435.

CHAN-PALAY, V., JONSSON, G. & PALAY, S. L. (1978). Serotonin andsubstance P coexist in neurons of the rat's central nervous system.Proceedings of the National Academy of Sciences of the USA 75,1582-1586.

DE KoNINCK, Y. & HENRY, J. L. (1991). Substance P-mediated slowexcitatory postsynaptic potential elicited in dorsal horn neurons invivo by noxious stimulation. Proceedings of the National Academyof Sciences of the USA 88, 11344-11348.

DE LANEROLLE, X C. & LAMOTTE, C. C. (1982). The morphologicalrelationships between substance P immunoreactive process andventral horn neurons in the human and monkey spinal cord. Journalof Comparative Neurology 207, 305-313.

ELLIOTT, P. & WALLIS, D. I. (1993). Glutamatergic and non-glutamatergic responses evoked in neonatal rat lumbar motoneuronson stimulation of the lateroventral spinal cord surface. Neuroscience56,189-197.

FRANCK, J., BRODIN, E., FRIED, K., RosEN, A., YAMAMOTO, Y. &FRIED, G. (1991). The effect of selective neurotoxin treatment onthe tachykinin levels in the rat ventral spinal cord. Neuroscience 45,339-345.

GARRET, C., CARRUETTE, A., FARDIN, V., MOUSSAOUI, S., PEYRONEL,J. F., BLAMCHARD, J. C. & LADURON, C. (1991). Pharmacologicalproperties of a potent and selective nonpeptide substance Pantagonist. Proceedings of the National Academy of Sciences of theUSA 88,10208-10212.

Guo, J.-Z., YOSHIOKA, K., YANAGISAWA, M., HosOKI, R.,HAGAN, R. M. & OTSUKA, M. (1993). Depression of primaryafferent-evoked responses by GR71251 in the isolated spinal cord ofthe newborn rat. British Journal of Pharmacology 110, 1142-1148.

HOKFELT, T., LJUNGDAHL, A., TERENIUS, L., ELDE, R. & NILSSON, G.(1978). Immunohistochemical evidence of substance P-like immuno-reactivity in some 5-hydroxytryptamine-containing neurons in therat central nervous system. Neuroscience 3, 517-538.

HOLSTEGE, J. C. & KuYPERS, H. G. J. M. (1987). Brainstem projectionsto spinal motoneurons: An update. Neuroscience 23, 809-821.

HosOKI, R., YANAGISAWA, M., GUO, J.-Z., YOSHIOKA, K.,MAEHARA, T. & OTSUKA, M. (1994). Effects of RP67580, atachykinin NK1 receptor antagonist, on a primary afferent- evokedresponse of ventral roots in the neonatal rat spinal cord. BritishJournal of Pharmacology 113, 1141-1146.

JACOBS, B. L. & AZMITIA, E. C. (1992). Structure and function of thebrain serotonergic system. Physiological Reviews 72, 165-229.

JESSELL, T., TsUNOO, A., KANAZAWA, I. & OTSUKA, M. (1979).Substance P: depletion in the dorsal horn of rat spinal cord aftersection of the peripheral processes of primary sensory neurons.Brain Research 168, 247-259.

796 T Kurihara, K. Yoshioka and M. Otsuka J Physiol.485.3

JOHANSSON, O., HOKFELT, T., PERNOW, B., JEFFCOATE, S. L., WHITE,N., STEINBUSCH, H. W. M., VERHOFSTAD, A. A. J., EMSON, P. C. &SPINDEL, E. (1981). Immunohistochemical support for threeputative transmitters in one neuron: coexistence of5-hydroxytryptamine, substance P- and thyrotropin releasinghormone-like immunoreactivity in medullary neurons projecting tothe spinal cord. Neuroscience 6, 1857-1881.

JOHNSON, S. M., KATAYAMA, Y., MORITA, K. & NORTH, R. A. (1981).Mediators of slow synaptic potentials in the myenteric plexus of theguinea-pig ileum. Journal of Physiology 320, 175-186.

KANAZAWA, I., SUTOO, D., OSHIMA, I. & SAITO, S. (1979). Effect oftransection on choline acetyltransferase, thyrotropin releasinghormone and substance P in the cat cervical spinal cord.Neuroscience Letters 13, 325-330.

KoNISHI, S. & SONG, S.-Y. (1987). Neurotransmission studied onisolated mammalian spinal cord. Japanese Journal ofNeuropsychopharmacology 9, 813-820.

KoNISHI, S., TSUNOO, A. & OrSUKA, M. (1979). Substance P andnoncholinergic excitatory synaptic transmission in guinea pigsympathetic ganglia. Proceedings of Japan Academy B, 55,525-530.

KURIHARA, T., YOSHIOKA, K. & OTSUKA, M. (1991). Involvement ofexcitatory amino acid receptors in the cutaneous nerve-evoked andsubstance P-evoked depolarization of rat motoneurons. JapaneseJournal of Pharmacology 55, suppl. 358P (abstract).

KURIHARA, T., YOSHIOKA, K. & OTSUKA, M. (1993). Involvement oftachykinins in the slow depolarization of neonatal rat spinalmotoneurones evoked by stimulation of descending pathway.Japanese Journal of Pharmacology 61, suppl. 243P (abstract).

MILLHORN, D. E., HOKFELT, T., VERHOFSTAD, A. A. J. &TERENIUS, L. (1989). Individual cells in the raphe nuclei of themedulla oblongata in rat that contain immunoreactivities for bothserotonin and enkephalin project to the spinal cord. ExperimentalBrain Research 75, 536-542.

NUSSBAUMER, J.-C., YANAGISAWA, M. & OTSUKA, M. (1989).Pharmacological properties of a C-fibre response evoked bysaphenous nerve stimulation in an isolated spinal cord-nervepreparation of the newborn rat. British Journal of Pharmacology98, 373-382.

OTSUKA, M. & YOSHIOKA, K. (1993). Neurotransmitter functions ofmammalian tachykinins. Physiological Reviews 73, 229-308.

PELLETIER, G., STEINBUSCH, H. W. M. & VERHOFSTAD, A. A. J.(1981). Immunoreactive substance P and serotonin present in thesame dense-core vesicles. Nature 293, 71-72.

RANDIC, M., Ryu, P. D. & URBAN, L. (1986). Effects of polyclonal andmonoclonal antibodies to substance P on slow excitatorytransmission in rat spinal dorsal horn. Brain Research 383, 15-27.

RoQuEs, B. P., FOURNIE'-ZALUSKI, M. C., SOROCA, E.,LECOMTE, J. M., MALFROY, B., LLORENS, C. & SCHWARTZ, J.-C.(1980). The enkephalinase inhibitor thiorphan shows anti-nociceptive activity in mice. Nature 288, 286-288.

SUZUE, T., YANAIHARA, N. & OTSUKA, M. (1981). Actions of vaso-pressin, gastrin releasing peptide and other peptides on neurons ofnewborn rat spinal cord in vitro. Neuroscience Letters 26, 137-142.

SUZUKI, H., YOSHIOKA, K., YANAGISAWA, M., URAYAMA, O.,KURIHARA, T., HosOKI, R., SAITO, K. & OTSUKA, M. (1994).Involvement of enzymatic degradation in the inactivation oftachykinin neurotransmitters in neonatal rat spinal cord. BritishJournal of Pharmacology 113, 310-316.

TAKAHASHI, T. (1985). Thyrotropin-releasing hormone mimicsdescending slow synaptic potentials in rat spinal motoneurons.Proceedings of the Royal SocietyB 225, 391-398.

TSUNOO, A., KoNISHI, S. & OSUKA, M. (1982). Substance P as anexcitatory transmitter of primary afferent neurons in guinea-pigsympathetic ganglia. Neuroscience 7, 2025-2037.

ULFHAKE, B., ARvIDssoN, U., CULLHEIM, S., HOKFELT, T.,BRODIN, E., VERHOFSTAD, A. & VISSER, T. (1987). Anultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonalboutons in the motor nucleus of spinal cord segments L7-S1 in theadult cat. Neuroscience 23, 917-929.

URBAN, L. & RANDIC, M. (1984). Slow excitatory transmission in ratdorsal horn: possible mediation by peptides. Brain Research 290,336-341.

VAccA, L. L., Hosms, J., ABRAHAMS, S. & NAFTCHI, E. (1982).Ultrastructural localization of substance P immunoreactivity in theventral horn of the rat spinal cord. Histochemistry 76, 33-49.

WALLIS, D. I. & Wu, J. (1993). The pharmacology of descendingresponses evoked by thoracic stimulation in the neonatal rat spinalcord in vitro. Naunyn-Schmiedeberg's Archives of Pharrmacology347, 643-651.

WARD, P., EWAN, G. B., JORDAN, C. C., IRELAND, S. J., HAGAN, R. M.& BROWN, J. R. (1990). Potent and highly selective neurokininantagonists. Journal of Medical Chemistry 33, 1848-1851.

YANAGISAWA, M. & OTSUKA, M. (1990). Pharmacological profile of atachykinin antagonist, spantide, as examined on rat spinalmotoneurones. British Journal of Pharmacology 100, 711-716.

YoMoNo, H. S., SUZUKI, H. & YOSHIOKA, K. (1992). Serotonergicfibers induce a long-lasting inhibition of monosynaptic reflex in theneonatal rat spinal cord. Neuroscience 47, 521-531.

AcknowledgementsThis work was supported by grants-in-aid for scientific researchfrom the Ministry of Education, Science and Culture, Japan (nos.04255101, 05557117 and 05454147). T. K. is a Research Fellow ofthe Japan Society for the Promotion of Science.

Received 28 June 1994; accepted 5 December 1994.