1 /M), and for the 8-receptor (D-Ala2-D-Leu6-enkephalin, 1-10 ,zM

J. Physiol. (1987), 391, pp. 141-167 141With 14 text-figuresPrinted in Great Britain

RELEASE OF SUBSTANCE P FROM THE CAT SPINAL CORD

BY VAY LIANG W. GO* AND TONY L. YAKSHtFrom the Divisions of Gastroenterology* and Neurosurgical Researcht, Mayo Clinic,

Rochester, MN 55905, U.S.A.

(Received 29 May 1986)

SUMMARY

1. The present experiments examine the physiology and pharmacology of therelease of substance P-like immunoreactivity (SP-l.i.), from the spinal cord in thehalothane-anaesthetized, artificially ventilated cat.

2. Resting release of SP-l.i. was 36+4 fmol/30 min (mean+S.E.; n = 106).Bilateral stimulation of the sciatic nerves at intensities which evoked activity infibres conducting at A/J conduction velocities (> 40 m/s), resulted in no change inblood pressure, pupil diameter or release of SP-l.i. Stimulation intensities whichactivate fibres conducting at velocities less than 2 m/s resulted in increased bloodpressure, miosis and elevated release of SP-l.i. (278+16% of control).

3. The relationship between nerve-stimulation frequency and release was mono-tonic up to approximately 20 Hz. Higher stimulation frequencies did not increase theamounts of SP-l.i. released. At 200 Hz there was a reduction.

4. Capsaicin (0-1 mM) increased the release of SP-l.i. from the spinal cord andresulted in an acute desensitization to subsequent nerve stimulation. This acuteeffect was not accompanied by a reduction in spinal levels of SP-l.i. measured 2 hafter stimulation.

5. Cold block of the cervical spinal cord resulted in an increase in the amounts ofSP-l.i. released by nerve stimulation.

6. Pre-treatment with intrathecal 5,6-dihydroxytryptamine (300 ,ug) 7 days priorto the experiment caused a reduction in the dorsal and ventral horn stores of SP-l.i.,but had no effect on the release of SP-l.i. evoked by nerve stimulation. Similar pre-treatment with intrathecal capsaicin (300 ,ug) resulted in depletion of SP-l.i. in thedorsal but not in the ventral horn of the spinal cord and diminished the release of SP-l.i. evoked by nerve stimulation.

7. Intense thermal stimulation of the flank resulted in small (20-35%), but reliableincreases in the release of SP-l.i. above control.

8. Putative agonists for the opioid ,u-receptor (morphine, 10-100 sM; sufentanil,1 /M), and for the 8-receptor (D-Ala2-D-Leu6-enkephalin, 1-10 ,zM; D-Pen2-D-Pen5-enkephalin, 10 uM), but not the K-receptor (U50488H, 100-1000,UM), produced adose-dependent, naloxone-reversible reduction of the evoked, but not of the restingrelease of SP-l.i. (-)-Naloxone, but not (+)-naloxone, resulted in a significant in-crease in evoked but not resting SP-l.i. release.

t To whom correspondence should be addressed.

V. L. W. GO AND T. L. YAKSH

9. Adrenergic agonists for the CX2-receptor (ST-91, 10-100 gM), but not for the al-receptor (methoxamine, 100 /LM) or f-receptor (isoprenaline, 100-1000 /M), pro-duced a significant reduction in the release evoked by nerve stimulation. This sup-pression was prevented by equimolar concentrations of phentolamine or yohimbine.5-Hydroxytryptamine (1000 /LM) was without effect. Muscimol (100 /M), but notbaclofen (100 ,CM), y-aminobutyric acid (GABA, 100-1000 ,SM) or glycine (100 /M),resulted in a small statistically significant reduction in the release of SP-l.i. evokedfrom spinal cord.

10. Tentative identification of the secreted immunoreactivity was established byshowing that the SP-l.i. in the perfusate collected during sciatic nerve stimulation orcapsaicin, co-eluted with authentic SP,-,, on both a reversed phase and Sephadex G-50 column.

11. Our studies suggest that stimulation results in repeatable and stimulus-dependent release of SP-l.i. from spinal cord. This effect may be mediated by small,probably unmyelinated, primary afferents and is under the inhibitory influence ofopioid (, and d) and a-adrenergic (a2) receptors.

INTRODUCTION

In the spinal grey, substance P (SP) is found primarily in two populations ofterminals: those arising from bulbospinal projections (H6kfelt, Ljungdahl, Stein-busch, Verhofstad, Nilsson, Brodin, Pernow & Goldstein, 1978) and those associatedwith primary afferents (Hokfelt, Kellerth, Nilsson & Pernow, 1975). Based on theimmunohistochemical localization of substance P in small (type B) ganglion cells, itappears likely that these afferent stores of SP are present in small myelinated orunmyelinated primary afferent fibres (Hokfelt et al. 1975; Tuchscherer & Seybold,1985). Electron microscopy and subcellular fractionation indicates that substance P-like immunoreactivity (SP-l.i.) is contained in synaptosomes and stored in dense corevesicles (Cuello, Jessell, Kanazawa & Iversen, 1977). Consistent with the localizationof SP in nerve terminals, it has been demonstrated that spinal stores of SP-l.i. aresecreted, in a Ca2+-dependent fashion, into the extracellular space in the presence ofdepolarizing concentrations of K+ (Jessell & Iversen, 1977; Mudge, Leeman &Fishbach, 1979; Akagi, Otsuka & Yanagisawa, 1980; Gamse, Lackner, Gamse &Leeman, 1981; Pang & Vasko, 1986; Yaksh, 1986). Subsequent in vivo investigationsdemonstrated that levels of SP-l.i. in the spinal extracellular fluid were increased bythe activation of slow but not rapidly conducting primary afferents (Yaksh, Jessell,Gamse, Mudge & Leeman, 1980). More recently, high-intensity thermal andmechanical stimuli have been shown to increase the extracellular levels of SP in thespinal space (Kuraishi, Hirota, Sato, Hino, Satoh & Takagi, 1985a; Duggan &Hendry, 1986). Similarly, direct stimulation of bulbospinal pathways will evokespinal SP release (Takano, Martin, Leeman & Loewy, 1984). These data demonstratethat spinal stores of SP exist which are subject to secretion in a manner ascribed toa neurotransmitter.

Because of the presence ofSP in at least two anatomically distinct pools, care mustbe taken to define the terminals from which the SP derives. The present paperdescribes studies which examine (1) the source of SP release after sciatic nerve

142

SPINAL SUBSTANCE P RELEASE

stimulation, (2) the physiological characteristics of that system and (3) the localeffects of selective agonists and antagonists on the release of SP-l.i. from spinalcord.

METHODSGeneral preparation

Cats (male or female; 2-2-3-6 kg) were anaesthetized by halothane inhalation. Once a surgicalplane of anaesthesia was reached, a tracheotomy was performed and femoral vein and arterycatheters placed. After the preparation was completed, the minimum motor threshold (m.m.t.: seebelow) was assessed and the animal given pancuronium (0-1 mg/kg); it was artificially respired bypositive-pressure ventilation with a mixture of 02: air: CO2 (1: 5: 0-02). Anaesthesia was maintainedwith halothane (0-8-1-0% inspired), which was held constant once preparations were completed.Mean arterial blood pressure (M.A.B.P.) and heart rate were recorded continuously. Arterial bloodgases were monitored periodically during the experiment using a model 113 Radiometer. Theventilation mixture resulted in blood gases which were: Pco ,35-38torr; Pa, 110-150 torr;pH: 7-35-7-41. Metabolic acidosis was corrected as needed wit; adequate bicarbonate infusions.A rectal probe was inserted and body temperature maintained at 36-5-37-5 °C with an under-

body heating pad.

Spinal perfusionTo establish perfusion of the caudal spinal cord, the cat was placed in a stereotaxic head holder.

A mid-line incision was made on the back of the skull and the cisternal membrane exposed by bluntdissection. A polyethylene catheter (PE-10, 0-6 mm o.d.: Clay Adams) for infusion of cerebrospinalfluid (CSF) was then passed 30-35 cm caudally to the level of the sacral cord. A second catheter(PE-90, 1-3 mm o.d.) for CSF withdrawal was then threaded over the PE-10 catheter in such a waythat the tip of the PE-90 catheter was located approximately 20 cm from the cisternal membraneat the approximate level of the thoracolumbar junction.For perfusion, the PE- 10 catheter was passed out of the PE-90 catheter through a small stab hole

in a piece of silicone tubing which was placed on the outside end of the PE-90 catheter. The PE-10 catheter was connected to the outflow channel of a two-channel peristaltic pump and the PE-90 catheter was connected to the inflow side of the second channel. The pump was calibrated toinfuse and withdraw nominally 100 ,ul/min. Because of variations in pump tubing this value mayhave varied by 10 ,ul/min between experiments, but calibration was carried out periodically duringthe experiment and the appropriate corrections were made. In several experiments, perfusion wascarried out by exposing the cauda equina and inserting a PE-IO catheter through a dural puncture.Outflow was collected as above.The medium employed for perfusion was artificial CSF (Tyce & Yaksh, 1981). The solution was

constantly bubbled with 95% 02 and 5% CO2. Osmolarity of this solution was 308-310 osm andthe pH was 7-41.

Cervical cold blockTo establish a reversible block oftransmission at the level ofthe cervical cord, the C1-C2 vertebrae

were exposed by a laminectomy extending laterally to the level of the root foramen. A U-shapedstainless-steel chamber 1 cm long, constructed to fit yoke-like was placed over the C1-C2 cord. Thelaminectomy space was then filled with agar. Temperature on the cord surface was measured witha thermister placed between the yoke and the cord. Under normal conditions, a water and alcoholmixture from a water bath maintained at 38 °C was circulated through the yoke. To establish alocal cooling of the cord, the circulating water and alcohol mixture from a bath maintained at -6to +8 °C was circulated as required. Under these circumstances, cord surface temperature wasmaintained at 4-8 'C. In one animal, a 24 gauge needle thermister (Yellow Springs Instruments)was inserted obliquely into the cat's cord during cooling. Temperature in this preparation aftercooling rose from 8 'C at the surface, to 11-13 'C as the cord was penetrated, to 16-19 'C 1-2 mmfrom the surface, and to 30-35 'C in the central cord (3-4 mm from the surface). This cooling wasfound to block the potentials recorded from the cord surface rostral to the block otherwise evokedby afferent stimulation. Re-perfusion of the chamber with a solution at 38 'C resulted in a returnto normothermia within 10 min and in a return of the evoked potential. Though not assessed, wefeel that it is not likely that conduction was blocked in the centre of the cord.

143

V. L. W. G0 AND T. L. YAKSH

Electrical stimulationAfferent stimulation was evoked by bilateral stimulation of the sciatic nerves. Pairs of hook

electrodes were placed just distal to the ischial notch after exposure of each nerve. A Grass S-88stimulator and SIU-5 constant-voltage units were employed. Unless otherwise indicated,stimulation was delivered in 1 s trains of monophasic pulses (20 Hz, 1 ms pulse width). The trainswere delivered repeatedly at the rate of 0 5 trains/s. To simplify the determination of the stimulusintensity required to activate small fibres, the voltage threshold necessary to produce a rapidtwitch of the distal limb with sciatic nerve stimulation was determined. This was done immediatelyprior to the administration of muscle relaxant.

Thermal stimulationTo assess the effects of high-intensity thermal stimulation on spinal SP release, thermal probes

(1 cm diameter) maintained at a temperature of 75 °C were applied bilaterally to the depilated skinof either flank; the probes were applied to the skin at the hind paw for 30 s and then movedproximally to an adjacent section of skin where the stimulation was repeated. This sequence wasrepeated continuously during the 30 min period of stimulation. In the non-anaesthetized catapplication of the probe has been found to evoke escape behaviour with a latency of 8-12 s (Yaksh,1978).Sural nerve recording

In a limited number of animals, recording from the sural nerve was carried out during sciaticnerve stimulation. To accomplish this, the sural nerve was exposed by dissection and the responsewas recorded in a monopolar fashion under oil with hook electrodes, using appropriate amplificationand averaging (Neurolog).Intrathecal catheterizationTo study the long-term effect of capsaicin or 5,6-dihydroxytryptamine administered into the

spinal space, cats under halothane (0-8 %) anaesthesia were prepared with a lumbar catheter (PE-10). This catheter was inserted intrathecally after exposure of the cisternal membrane as previouslydescribed (Yaksh, 1978). Aseptic surgical procedures were used and the animals received 300000units penicillin pre-operatively.

Sample collectionPerfusion samples for assay were collected on ice in a polyethylene test-tube which contained

0-1 ml 1 M-acetic acid. Upon collection, samples were frozen on dry ice and lyophilized with aSavant spinvac unit. For tissue levels, frozen tissue samples were weighed and homogenized in0-1 M-HCI to yield a tissue concentration of 50 mg/ml. Aliquots of the supernatants were taken forassay.

Assay proceduresSubstance P. Antiserum no. 4892 obtained from immunized rabbits was employed. SP fragments

possessing the amino terminal showed a high degree of cross-reactivity. Peptides that showed nocross-reactivity at 100 ng/ml included bombesin, CCK-8, neurotensin, somatostatin, met-enkepha-lin, vasoactive intestinal polypeptide (VIP) and peptide-histidine-isoleucine (PHI: a twenty-seven-amino-acid peptide with a close sequence homology to VIP).

Fig. 1 presents the antiserum displacement curves carried out with a series of SP fragments. Asindicated, significant cross-reactivity remains for fragments from 1-11 to 6-11 and from 1-11 to1-9. Further reduction in fragment size from either the amino terminal or the carboxy terminalresults in essentially complete loss of reactivity. Removal of the carboxy terminus reduces cross-reactivity somewhat. A second tachykinin identified in spinal cord, substance K (SK), has anIC60 (the concentration giving 50% of maximal inhibition) which is 100 times that of SP. Tyr8-SP,SP,-,, and the oxidized form of SP cross-react completely.The ligand, Tyr8-SP was iodinated by a modified Greenwood-Hunter method (Greenwood,

Hunter & Glover, 1963) using Na 126I. The reaction was stopped by the addition of sodiummetabisulphite and the unreacted label initially separated using a cellulose CF-I column and elutedwith 0-2 M-glycine (pH 8-1) and acidified plasma. 125I-Tyr8-SP was further purified by gel chroma-

144

SPINAL SUBSTANCE P RELEASE 145

tography (Biogel P-I0). The ligand was diluted to 5000 counts/min per 01 ml in assay buffer (4%v/v) rabbit serum, 004 M-KH2PO4, 1% (w/v) BSA, 0 01 M-EDTA, 2% polyethylene glycol-8000(PEG-8000) and 002% thimerosal; pH '55). For assay, lyophilized samples or authentic SP werereconstituted in assay buffer. The reconstituted sample was added to polystyrene assay tubes(12 x 75 mm) and incubated with 0lml antisera (1: 15000) and 07 ml buffer. After a 16 hincubation at 4 °C, "261-Tyr8-SP (01 ml) was added and incubated for an additional 24 h. Thebound complex was precipitated by adding a burro anti-rabbit serum and then centrifuged. Thelower limit of detection was 1-0 pg SP (0 7 fmol)/tube. Inter- and intra-assay variation were 11 and5% respectively. All assay values were calculated with standard curves made up from the perfusateto be assayed.

1 11Arg-Pro-Lys-Pro-Gin-Gin-Phe-Phe-G Iy-Leu-Met-N H2

100 ~_ _

8/o0' N SP fragment1 9-112 8-11

60 3 7-11B/Be 4 6-11(% 5 5-11

40 - ~~~~~~~~64-11\ \\ \\ 17 3-11

8 2-11

20 991-11

0 \

100 ~~~~~~~~~~~~~~10Eledoisin10 11 SK

N N 12 1-780" 13 1-9N. ~~~~~~~~~~~14Free acid11 15 1-11

60~ 12 16 Tyr8-SP/BIB 17 Physalaemin(%)

r ~ 15_16

p- p- I I I ails I I ,,,,,,1710 100 1

Concentration (pg/mi)

1314

* . I * LLW1000 10000

Fig. 1. Ligand displacement curves carried out with a variety of substance P (SP)fragments using antiserum no. 4892. SK, substance K. B/BO, percentage bound.

Neurotensin. Levels of neurotensin were assessed in spinal cord using antiserum no. N398. Thisantiserum recognized the amino terminal. Details of the assay are given elsewhere (Yaksh,Schmauss, Micevych, Abay & Go, 1982). The absolute sensitivity of the assay is 3 pg/tube. Inter-and intra-assay variation is 10 and 4% respectively.

VIP. This peptide was assayed in spinal cord using antiserum no. 4823. Details of this assay are

V. L. W. GOAND T. L. YAKSH

given elsewhere (Yaksh, Abay & Go, 1982). The antiserum recognizes the carboxy terminal.Absolute assay sensitivity is 3 pg/tube. Inter- and intra-assay variation is 15 and 7%, respectively.

5-Hydroxytryptamine and noradrenaline. These amines were extracted from tissue samples,deproteinated and separated on a CG-50 column. The levels of amines in the concentrated eluatewere assessed by high-performance liquid chromatography (h.p.l.c.) using electrochemical detec-tion. Details of the extraction and the assay are given elsewhere (Howe, Yaksh & Tyce, 1987). Theabsolute sensitivity of the h.p.l.c. assay for both 5-hydroxytryptamine and noradrenaline was25 pg/sample. Routinely this method is adequate for detection of both amines in less than 50 mg oftissue.

Characterization of immunoreactivityTo characterize the ligand recognized by the antisera; three procedures were employed. First,

pooled samples collected during release were lyophilized and injected onto a reversed-phase h.p.l.c.column (LKB Instruments). Immunoreactivity was eluted with a linear gradient of acetonitrile(10-60% in phosphate buffer). The profile of SP-l.i. elution was compared with that obtained withauthentic standards. Secondly, other samples were pooled and after lyophilization were taken upin 1-5 ml phosphate buffer (001 M, pH 7 6) containing 0-5% bovine serum albumin. 1 ml sampleswere placed on a 1 x 100 cm Sephadex G-50 (superfine) column at 4°C and eluted at 0-8 ml/min.Columns were equilibrated and eluted with 0 01 M-phosphate buffer. Blue dextran 2000 and Na 1251iodide were used to identify the void volume and the salt peak, respectively. Thirdly, liganddisplacement curves were carried out with serial dilution of pooled perfusate samples and theirslopes compared to the slopes of dilution curves obtained with authentic SP.

DrugsAll peptides are obtained from Peninsula Laboratories (Belmont, CA, U.S.A.). All concentrations

of standard were corrected for true amino acid content. All solutions were prepared from analyticalgrade products. Peptide standards for radioimmunoassay were verified by chromatography.Capsaicin and 5,6-dihydroxytryptamine are from Sigma (St. Louis, MO, U.S.A.).

Drug administrationAgents were administered into the spinal space by their addition to the spinal superfusate. The

following agents were examined: baclofen (Ciba-Geigy); DADL (D-Ala2-D-Leu5)enkephalin, Bur-roughs-Wellcome); DPDPE (D-Pen2-D-Pen6)enkephalin,courtesy of Dr Victor Hruby,Tucson, AZ,U.S.A. and Peninsula Laboratories); GABA (y-aminobutyric acid, Sigma); glycine (Sigma);isoprenaline (Riker Laboratories); methoxamine HCl (Burroughs-Wellcome); (-)-morphinesulphate (Merck); muscimol (Ciba-Geigy); (-)-naloxone HCl (Endo); (+)-morphine and (+)-naloxone HCl (courtesy of Dr Dick Hawk, National Institute on Drug Abuse); phentolamine HCl(Ciba-Geigy); ST-91 ((2-[2,6-diethylphenylamino]-2-imidazoline), Boehringer Ingelheim); sufen-tanil (Janssen); U50488H (trans-( + )-3,4-dichloro-N-methyl-N[2-91-(pyrrolidinyl)cyclo-hexyl]benzacetamide methane sulphonate, Upjohn); and yohimbine HCI (Sigma).

StatistiCs

All results are presentedas the mean +S.E. of mean. For analysis of release due to stimulation,the levels of SP-l.i. in the perfusion fluid are presented as a percentage of the levels measuredimmediately prior to stimulation which is the percentage of basic release. Student's t test for pairedsamples was used to determine its statistical significance. Multiple comparisons between groups

were carried out with a one-way analysis of variance. If significant, the ordering of significance was

assessed using a Newman-Keuls analysis (Winer, 1962).

RESULTS

Spinal relea8e of SP-l.i. by afferentstimulationBilateral stimulation of the sciatic nerves at intensities which evoke activity

conducting at 40-100m/s (3 x minimum motor threshold: m.m.t.) failed to alter thelevels of SP-l.i. ih superfusates of spinal cord. In contrast, elevation of stimulation

146


intensities, to 30 x m.m.t. in order to activate fibres which conduct at velocities of1-2 m/s, resulted in highly significant increases in levels of SP-l.i. in spinal super-fusate. Fig. 2 (left), presents a representative experiment displaying this effect withan individual cat. Aside from the effect on SP-l.i. release, low-intensity stimulationof A/ fibres failed to have any effect on either heart rate or pupil diameter. Incontrast, high-intensity stimulation of A/f, Ad and C fibres resulted in increases ofboth systolic and diastolic blood pressures as well as an increase of pupillary diameter(Fig. 2).

200] 200 AO

E 160~~~~~~~~~~~

1200 4A3/A/C

400

120 W c

Etl@016000~ ~ ~ , A/AOCA reeas

10 A0 140040 30

80 *300

C60-E0 200-0~~~~~~~~~~~~~

E 40

Un 20 10

AOAO/A5/C ~~~~~~~B.P. Pupil SP-I.i.At3A13/A6/C ~~~~~~~~~~~~~~release

0

0 60 120 240 300Time (min)

Fig. 2. Left, single experiment on one cat displaying the effects of stimulating the sciaticnerve at intensities evoking Afl or A/?/A&/C fibre activity on the levels of substance P-likeimmunoreactivity (SP-l.i.) in spinal superfusate and the concurrent changes in systolicand diastolic arterial blood pressures and pupil diameter as a function of time. Right,summary of the results from six experiments showing the changes (percentage of baseline) in mean arterial blood pressure (B.P.), pupil diameter and SP-l.i. in the spinalsuperfusates after evoking A,@ (top) and Af/A8/C fibre activity (bottom), in halothane-anaesthetized cats. Details of the preparation are described in the text. *P < 0 05 ascompared to 100.

Repeatability of release. As shown in Fig. 3, repeated bilateral stimulation resultedin significant increases in SP-l.i. in three stimulation intervals separated by 1 h.Statistical comparison reveals that for pupil size, mean arterial blood pressure andSP-l.i. release, stimulation at all three intervals, resulted in levels which were greaterthan the immediately preceding control level (t values = 3-16-4-51; P < 0-05); thethree responses were not different from each other when compared using a one-wayanalysis of variance (f = 1P41-1-75; d.f. = 2/5; P > 0 20).

148 V. L. W. GO AND T. L. YAKSH

Correlation between autonomic response and SP-l.i. release. The increases in spinalSP-l.i. release are correlated with the changes in M.A.B.P. evoked by stimulation ofsciatic nerves. This is emphasized by Fig. 4 which presents the relationship betweenthe changes in spinal SP-l.i. release and M.A.B.P. after high-intensity stimulation infifteen animals.

8

6 400 - Pupil

180 2 C 300

I~~~~~~~~~~~~~~EE 140

co C: ~~~~100IL7iD100 2 L''BP

0 T*

80~~~~~~~~~~~gT*O.LTiW

80 C:~~~~~~~~~~~~~~~~~~0

a 60 SP0~~~~~~~~~~~~~~~0ET

4020

20- A- -

A B C0 I I £ ~~~~~~~~~~~~~ ~Stimulation

0 60 120 180 240 300Time (min)

Fig. 3. Left, the effects in a single cat of three repetitive stimulations of the sciatic nerveat Afl/M/C fibre intensity on the release of SP-L~i. into the spinal superfusate, arterialblood pressure and pupil diameter. Right, histograms present the results as percentage ofbase-line levels in four animals in each of three repetitive stimulation intervals on pupildiameter (top), M.A.B.P. (middle) and SP-l.i. release from the spinal cord (bottom). *P <005 as compared to 100.

To determine whether the increase in SP-l.i. release is associated with the changesin arterial pressure, methoxamine (3-15 ,sg/kg, i.v.), was infused to produce a stableelevation in M.A.B.P. for at least 25 min during the appropriate collection intervals.In four experiments, increases in M.A.B.P. comparable to those observed during sciaticnerve stimulation were not associated with any significant change in SP-l.i. releasedfrom spinal cord (Fig. 4).

Physiological characteristics of spinal systems from which SP-l.i. is released. Forthe spinal systems which release SP-l.i. the afferent stimulation threshold, the fre-quency dependency and the apparent refractory period were assessed.To obtain a physiological index of stimulation intensity, we sought to establish the

relationship between SP-l.i. release and the threshold voltage (m.m.t.) required toproduce a contraction of either hind limb just prior to injection of muscle relaxant.


In three cats, three sequential stimulations were carried out at 1 h intervals at pre-determined multiples of the m.m.t. At 3 x m.m.t. a rapidly conducting potential(> 40 m/s) was seen. At 10 x m.m.t. a compound potential, the slowest discerniblecomponent of which travels at around 10-15 m/s, was observed. At the higheststimulation intensity of 30 x m.m.t., a complex wave was noted having a small poten-tial which travelled at less than 1-2 m/s. As indicated, increases in SP-l.i. levels werereliably observed at 30 x m.m.t. (Fig. 2). This 30 x m.m.t. was used in all subsequentexperiments unless otherwise stated.

300 0 *

0 0

en _

0~~~~~~~~~~~

_0 * *

0

1200 *

a) 0

10 00 A

100 150 200M.A.B.P. (% of base line)

Fig. 4. The levels of SP-l.i. release (percentage of base line) is plotted against the con-current change in M.A.B.P. (percentage of base line) observed during stimulation of sciaticnerves at intensities resulting in the activation of either A, (0), or afl/A8/C fibres (@).Animals in which no stimulation was carried out, but methoxamine (15 jug/kg) was infusedintra-arterially to produce corresponding elevations in blood pressure, are also shown(A).

To determine whether stimulated release was frequency dependent, animals wereprepared as described above and stimulated at 30 x m.m.t. at frequencies of 2-200Hz (Fig. 5). The frequency release curve is not monotonic. Maximum SP-l.i. releasewas observed at 20 and 50 Hz, whereas at stimulation frequencies of 200 Hz therewas a reduction in release in the four experiments.To assess the apparent relative refractory period ofthe spinal systems which release

SP-l.i., sciatic nerves were stimulated at 30 x m.m.t. at 20 Hz, with the pulsesarranged in pairs. With intervals between pulses in each pair being 50, 10, 2 or 0 ms.Thus, the 50 and 0 ms intervals are equivalent to stimulating at 20 and 10 Hz,respectively. After 1 h, a second stimulation sequence was applied at 30 x m.m.t. at20 Hz with 50 ms interpulse intervals. Fig. 6 (left) shows the release evoked in asingle experiment using this paradigm. Fig. 6 (right) presents the summary of elevensuch experiments carried out with different intervals between pulses. There were nodifferences in amount of SP-l.i. released with 0, 2 and 10 ms interpulse intervals. Themagnitude of the increase of SP-l.i. released at an interpulse interval of 10 ms,however, is significantly less than that released with a 50 ms interpulse interval

coa,.0

4-

0

C,,

V. L.W.JGO AND T. L. YAKSH

0

0

300k

0

200p-

100o

.0

0

.

00

2 5 10 20 50 200

Stimulation frequency (Hz)

Fig. 5. SP-l.i. release (percentage of base line) is plotted vS. the frequency at which thesciatic nerves were stimulated bilaterally at 30 times the minimum motor threshold(m.m.t.). Each point represents a single experiment. The line connects the means of theresponses at each frequency.

0

300[._

( 2.0

0

MS 200

C,,

'0 Hz 20 Hz

2ms 50ms

0 1 2 3 4 5Time (h)

I

I

:* 0

0

0

.0

100-

02 10 50Double-pulse interval (ms)

Fig. 6. Left, results showing the release of SP-l.i. from the spinal cord in a single animal.At the time indicated by the black bar the animal received bilateral stimulation of thesciatic nerves at 20 Hz (30 x m.m.t.). In the first stimulation sequence the interval betweenpulse periods was 2 ms, while in the second stimulation sequence the inter-pulse intervalwas 50 ms. Right, SP-l.i. levels in spinal superfusates (percentage of base line) observedduring double-pulse stimulation at intervals of 0, 2, 10 and 50 ms. The experimentalparadigm for these data are as presented in the Figure on the left. All animals receiveda first stimulation sequence of 20 Hz (30 x m.m.t.) in which the intra-pulse pair intervalswas 0, 2, 10 or 50 ms. In the second stimulation sequence, the animal received 20 Hzstimulation (30 x m.m.t.) with an intra-pulse pair intervals of 50 ms.

150

100

E0

0E

C,,

60

20


(180 vs. 260% of base line; P < 0-05). As emphasized in Fig. 2, the changes in releasewith repeated stimulation are minimal when stimulus parameters are heldconstant.In one cat, the area under the compound action potential was measured at the

dorsal roots after laminectomy, using a similar stimulation and recording paradigm.The voltage associated with the fibre population conducting at velocities of 2 m/s orless was reduced to 14 + 9% of the control value with a 10 ms interpulse interval andwas essentially occluded with a 2 ms interval. More rapidly conducting componentswere not significantly diminished by a 10 ms interpulse interval (89+ 7% of control).

Manipulation of the content of spinal systens which contain SP-l.i.In this series of experiments, the effects of cervical cord blockade or intrathecal

capsaicin or 5,6-dihydroxytryptamine were examined on the afferent-evoked releaseof SP-l.i.

Cold block of cervical cord. Fig. 7 presents the effects of a cold block of the cervicalcord on the levels of SP-l.i. in the spinal superfusate evoked by stimulation at30 x m.m.t. This cold block results in a statistically significant (P < 0-05) increase inspinal SP-l.i. release and a blockade of the miosis.

Capsaicin-evoked release and desensitization. Capsaicin has been shown to interactwith populations of small lightly myelinated or unmyelinated primary afferents(Fitzgerald, 1983) and to lower the levels of spinal cord SP-l.i. in the cat (Abay,1982).

Capsaicin (0-1 mM) added to the spinal perfusate results in a marked increase inSP-l.i. release and an elevation in blood pressure and pupil diameter. Nerve stimu-lation (30 x m.m.t.) after capsaicin fails to increase the slightly raised levels of SP-l.i.in the spinal superfusate (Fig. 8). A significant attenuation of the changes in pupildiameter and blood pressure evoked by the subsequent sciatic nerve stimulation isalso observed.To determine whether the failure of release after capsaicin reflects an acute deple-

tion of SP-l.i., spinal cords were collected immediately at the end of an experiment,approximately 120 min after termination of capsaicin perfusion. Measurement ofdorsal horn SP-l.i. levels in a cat shortly after the administration of intrathecalcapsaicin revealed no statistically significant change in the levels of SP-l.i. as com-pared to control animals (148+ 15 pg/g; n = 5 vs. 171 + 18 pg/g; n = 6, respectively;P > 0 20). Thus, the failure of stimulation to evoke release appears to occur in theabsence of any change in the over-all dorsal horn content of SP-l.i.

In earlier experiments pre-treatment with a single injection ofintrathecal capsaicin7 days prior to killing produced a fall in spinal levels of SP-l.i. (Abay, 1982). Todetermine whether such a depletion has any effect on release, cats under halothaneanaesthesia were prepared with an indwelling intrathecal catheter and received anintrathecal injection of either 300 jug capsaicin (0-3 ml) or vehicle (50% dimethyl-sulphoxide and saline, 0-3 ml). After 2 h, the anaesthesia was stopped and theanimal allowed to recover. 7 days later the animal was anaesthetized and the releaseof SP-l.i. was measured. Immediately after completion of the stimulation paradigmas used for acute capsaicin treatment (see Fig. 8), the animal was killed and the levelsof 5-hydroxytryptamine, VIP, neurotensin and SP-l.i. were determined in the dorsaland ventral lumbosacral spinal cords.

151

152 V. L. W. 0O AND T. L. YAKSH

Following surgery, vehicle-treated animals displayed no untoward signs. However,during the first 2 days all capsaicin-treated animals displayed clear signs of hind-limbataxia which gradually diminished during the ensuing period. In addition, allcapsaicin-treated animals displayed urinary incontinence: their bladders wereemptied twice daily by abdominal compression. On autopsy, all capsaicin-treatedanimals had significantly enlarged bladders.

400 Pupil

180 3n00f-

A.

E 140 100CLM.A.B.P.

0 20060 *100 1m [T1n80~~~~~~~~~~~~

Fig. . Lef, result n a inglecatshowig th relese o SP-li., he chngesinPsytoli

0)cm

o 60 3(00l0E

40 -200

of~~2-hpnlcr oe a)S = stmlain Riht Iitgaspeettema eut

20~~~~~~~~1 1

Cold block s s= +

in the resece ( + cld lock of ervial cld lock *P 0 0 as colId block0 60 120 180 240

Time (min)

Fig. 7. Left, results in a single cat, showing the release of SP-l., the changes in systolicand diastolic blood pressure, and pupil diameter as a function of stimulating the sciaticnerves at 30 x m.m.t. (black bar) before and during cold block applied at the C1-02 levelof the spinal cord (open bar). S = stimulation. Right, histograms present the mean resultsin six cats, for the change in pupil size, in M.A.B.P. blood pressure and in the release of SP-

Lsi. in spinal superfusates (percentage of base line after stimulation) in the absence (S) andin the presence (S + cold block) of cervical cold block. *P < 0-05 as compared to 100.

Capsaicin resulted in a significant reduction in dorsal horn levels of SP-L~i. whencompared to vehicle-treated controls (Table 1). Neither the ventral horn levels of SP-lU. nor of 5-hydroxytryptamine were affected. The levels of VIP and neurotensinimmunoreactivities in the dorsal or ventral horns were not affected. However, inanimals pre-treated with capsaicin the release of SP-l1i. evoked by sciatic nervestimulation or by the superfused capsaicin was almost abolished (Table 1).

Effects of intrathecal 5,6-dihydroxytryptamine. To examine the effects of destroyingspinal 5-hydroxytryptamine terminals on evoked SP-l.i. release, cats under halo-thane anaesthesia were prepared with a lumbar intrathecal catheter and received anintrathecal injection of 5,6-dihydroxytryptamine (300 ,ug/0.2 ml saline). After 7


e~~~~~~~~ N .

- ~ ~ ~ *C CC-.L

o~~~~~~~r

00~~~~~

o~~~~ CO

*~~~~~--

~+I +1 +1 +1G)CO 0 CON

- N -> I)>- -~ C 1+l +l +l +l .

, 0 O CO CX 10

++1 +1 +1 +1

¢ +l +z es eq F

*00 00 CDE-1~ ~ ~~~0

Cs CL COC

+1 +1 +1 +1 30 rC O

10 10 - 10 b01 0 1 "0m

* 1 01v o oI01 1 IIP -_ > 11* 10 Ci Hv +1 +1 +1 +1 .CLx 10txo V - . ^

¢+1 +1-I--- -~~~~o-4+1 ++ I,

16 ~ 01 0+1 CO 1 11

01 01

+1 +1 +1 +l

00es _4> s S

* * V- vo 4-

Aq- - ,- ,

v f <s~~a c, _ 1c

00

~ '~4 C '~I4 go -P Cb 0

o0

004~ *> H 4D

153

= D

.0(1O ," .1-

4a'

Ca

0-

-4a9

^

CLO)

CL*0 .

X CL

._ 0e-

fw

o 8

0

,, ._

00C

04o

-0

X

; .

10

00

*-

00* -4

-

ca

,

F-4Q

4)

E,0

I=ca


days, the sequence employed with sciatic nerve stimulation and capsaicin (01 mm)(Fig. 8) was carried out. The animals were then killed and the levels of SP-l.i., 5-hydroxytryptamine and noradrenaline were assessed in the dorsal and ventral horns.While 5,6-dihyroxytryptamine resulted in a significant reduction of 5-hydroxy-tryptamine in the dorsal and ventral horns, the levels of SP-l.i. in the dorsal hornswere reduced by approximately 20% and those in the ventral horn by about 70%(Table 1). There were no significant changes in the levels of noradrenaline. Both

I 700L~~~E Pupi~

jE 700(-3000

180 -T

E 140- LwH

400

20 s-i CAP S-2 20

-- 100~~~~~~~~~~~~S-iCAP 5-2

.~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~( M. A. B.IP.jj l

0 60 120 180 240 300Time (min)

Fig. 8. Left, the results in a single cat, for SP-l.i. release and the corresponding changesin systolic and diastolic blood pressure and pupil size as a result of bilateral stimulationof the sciatic nerves at 30 x m.m.t. before (S-i) and after (S-2), the administration ofcapsaicin (100 AM) (CAP) into the spinal superusate. Right, the mean results in seven catsof the effects of S-i, capsaicin perfusion and S-2. *P < 0~05 as compared to the pre-stimulation values.

electrical stimulation (30 x m.m.t.) and capsaicin (0 1 mM) evoked significant in-creases in the levels of SP-l.i. in the spinal superfusate which were not different fromthose in animals treated with vehicle without 5,6-dihydroxytryptamine.Animals treated with 5,6-dihydroxytryptamine showed no evident signs of be-

havioural or motor disabilities.

Effects of thermal stimulation on spinal SP-l.i. releaseIntense bilateral thermal stimulation of the skin resulted in small increases in SP-

l.i. levels in the perfusate of all five anaesthetized animals (Fig. 9). The mean restinglevel in these animals was 32+8 pg/30 min. The mean increase observed during the


time of thermal stimulation was 12 + 3 pg/30 min, a 38% increase over base line.Significantly, in contrast to control experiments, after thermal stimulation there wasa progressive increase in the basal release of SP-l.i. (19± 3 pg/30 min, e.g. a 58%increase above pre-stimulated control).

In two animals, control experiments were carried out in which probes at roomtemperature were applied to the flank. No change in the resting levels of SP-l.i. wasobserved (+ 3 and -4 pg/30 min, respectively).

50

301150

30

.'550

cn ,

20 6 140

20r

0 60 120 180 240Time (min)

Fig. 9. Levels of SP-l.i. found in the spinal superfusate before and after the applicationof a 75 °C thermal stimulus to the flank of the anaesthetized animal at the time indicatedby the black bar (see text for details of stimulation). Each record represents a singleanimal.

Effects of spinally administered receptor agonists and antagonistsIn the following sections, the effects of ligands for opioid, adrenergic, 5-hydroxy-

tryptaminergic and inhibitory amino-acid receptors on the spinal release of SP-l.i.during resting and stimulated conditions are described.

Opioid agonists. The superfusion of morphine sulphate (100 ,tM) under non-stimu-lated conditions had no effect on the levels of SP-LI in spinal perfusate or bloodpressure. However, morphine attenuated the release of SP-l.i. and the autonomicresponse otherwise evoked by high-intensity (30 x m.m.t.) stimulation of the sciaticnerve (Fig. 10). This effect is concentration dependent over the range of 10-100 suM-morphine.

In a single experiment, the effects of (+)-morphine (100 UM) were examined. Incontrast to (-)-morphine sulphate, it had no effect on SP-l.i.-evoked release. Therelease evoked in the presence of (+)-morphine was 306% of the pre-stimulationvalue.

155


The effects of morphine were antagonized by an equimolar concentration of nal-oxone (100,M) in the perfusate (see Fig. 11). To determine whether the effects ofnaloxone reflected a stereospecific interaction, equimolar concentrations of (+)-naloxone HCl (100 ,lM) were examined in two experiments in the presence of mor-phine (100,M). The release of SP-l.i. remained depressed in the presence of (+)-naloxone (48 and 31% of the control stimulation values, respectively).

40 Pupil*

16 -S 2 300 0E 3

120 6

i~IM.A.B.P80-

'200 ~ *0)

80-

60 -

-~~~~~~~~ 2~~~~~~00A

and~~~~~O 100 ie ttetme niae ytebas --opie(OR 0 l)ad()naloxone~ ~ ~~~~NA(NL+0U)wr de otesia spruae ih,hsorm rsn

0

0nthe resenc 60 120phine (180 240M)300ne -

Fig 10. Leftcopresults inoaesingleat, meshowing ntheeffeceofeblatealystiulaio (30oxt.m.t

20rphz)e, catuhstmsiniaed bythecioblcian theevkdrlevselof SP-l.i.i27ndthe%spnasuperfusate2 andthe%conrrn changeslduingarteiluytoiandidiastotioboorepressurely.I

andtpupilcsize, Atthe timsesuintdmicste ath b (-)-morphine(MOR,100,UM) anld(

neeraloxn (NAL, 100rsso ofwereoadedtotespnleasuerfsae Righthistoogramus poreSent.ithele meawresoe for sixcas6 prcntgofbavluse lineevntokel bstimulation,repcarivedlouC~aomparedbtoexevokedtwreles measuried intwthe -absenceofanydrug(cnt.).L)

Intwot exprpimntsthadiionftedrcporaoitsufentanil,D L(1-OU)inM loedpnetfsike,

Fmorpinished,cause ar in in thereleaseofSP-l.i.ote(26 ad 2%f.1

controluvs.a12 and 202%cofrrn coantrol duingdrugilytoiadminiastration,oorepretively.Ieitherpicsie,Athesubsequentdministrabytion ofrs (- )-naoxohne(MO,100#tm)resltd ireeraloxofeNLthisspprsso ofr evokedrotespnleasue.fuaTheRghpohst-aooneralus foresP-lnireeae weanres301onde 261%ias(pretgofthbvlusefocontreolebstimulation,repctrively.uComparalexmpaerimoentse wrelescearuriedout wthe -Aab-D-Leuo-enkephaugn(DADL).

As twixeimnsthemrhnnsufentaonil, DADL (1-10eceM)toiagdonse-depfendenil(tmfashion,

diiihdtereleasewofSP-lnd26i.oftherwluse evokebyonervestimulation, (Fecigel.1)


0

00

0 0

0

0

S

0

0

DADL DADL DADL1 10 10

+

.

S

0

0~~~~

0

S~~~~

0

DPDE U U (-)NAL (+)NAL10 100 1000 100 100

NAL100

Intrathecal drug concentration (pM)

Fig. 11. The effects of morphine (MOR), D-Ala2-D-Leu5-enkephalin (DADL), U50488H(U), D-Pen2-D-Pen5-enkephalin (DPDE), (-)- and (+ )-naloxone (NAL) on the release ofSP-l.i. evoked by sciatic nerve stimulation. In each case the paradigm shown in Fig. 10was used. The results are expressed as the release observed during sciatic nervestimulation in the presence of the agonist divided by the base-line release x 100. Each dotrepresents a single experiment.

0

S

0 :* S

0 0

* 0 a

00

SS

0

: 0

1-:-0-

0

0

ISO ISO MET MET ST ST ST/P 5-HT5-HT P

100 1000 100 1000 10 100 100 100 1000 100I ntrathecal drug concentration (#M)

Fig. 12. The effects ofisoprenaline (ISO), methoxamine (MET), ST-91 (ST), and 5-hydroxy-tryptamine (5-HT) on the release of SP-l.i. evoked by sciatic nerve stimulation. Detailsof the experiments are as described in Fig. 11. P, Phentolamine.

This inhibitory effect was reversed by the subsequent addition of naloxone (100 ,UM)to the superfusate. D-Pen2-D-Pen5-enkephalin, a highly selective 6-receptor ligand(10 UM), also resulted in a dimunition of release of SP-l.i. evoked by stimulation(Fig. 12).

150 r

157

I0

E

-6

0R0

C,,tn

100

50

0

0.

0

0

MOR MOR10 100

MOR100+NAL100

150w

looF

S

---* 0

C0

'._

ECa

-a

08-40

CLIcn

0

0

50-

O0

01

158 V. L. W. G0 AND T. L. YAKSH

U50488H, a K-receptor ligand, had no effect at concentrations up to 1000,UM.Higher concentrations could not be used because of interference with the assaystandard curve.

6 400 PUPil4E

160E 300

,0.

CL

80 181 00-

c

80

timeidctdbtebakb(3xm..,2HzothreesoSPli,hsyti200

20~~~~~~~~~~~~~~

indiatebytheblak brs,ST-91 (S,1010ad0hnoaie(HE,10z)wr

presence ~ ~ ~ ~~HECoftST-9,9o ST-91anphnoaie*P<05acmardttebs-le

80

MC' 600 0 20 1802000

time indiated bythebo-tmlack dbre(3exaint, 20oHzonthe releases (ofrphine,tesysetolic,DAnDLd, tliloD-e Dpressurenkpa,U08Haandpuildimecrinaain leexprien. ctthe tiestnrladdmesuedtoth spinal superfusate.Rg T, eresult lvesforfive cats,sshowinnhffcso SP1in

release, meange artrial6+bloo prssr andpupil diamether prduedbysimulationainthanewapreene ofdition orST-9-nalxndpento00laMine *requent5 aesucopaed to tiherbase-line

evkdS-..release.ta bevdi h oto tiuainpro Fg1.I

Wiarth regaerdmeto,no-stimuatooedreleaseM,noeowhen agonisse (morphie,asufcentanior,DADLe,D-ew D-eaekehsnU5i48oH)ha anygdetectabl effect onrestingreas(5+7v.58+9p/ml;

found with 100 /~~~M-morphine (12 ± 6 %)0.

P> 010, paired t test), but resulted in an increase in release of SP-l.i. evoked byhigh-intensity stimulation (P < 0-05; Fig. 11). To determine whether this effect ofnaloxone was mediated by an opioid receptor, three experiments were carried out


with (+)-naloxone HCl at equimolar concentrations (100,M). Addition of (+)-naloxone had no effect on resting release and did not increase release of SP-l.i. bystimulation (see Fig. 11).

Adrenergic and serotonergic agonists. The addition of the ,f-agonist isoprenaline, theal-agonist methoxamine or 5-hyroxytryptamine at the highest concentrations which

0 O

50

10600

SP-l.i.(pg/ml)

0100

50

0

Control

Aj3/AS/C

Capsaicin

30Retention time

60

Fig. 14. Elution profile of SP-l.i. using an acetonitrile gradient on an h.p.l.c. column. Thematerial was obtained from pooled perfusates collected before stimulation or after Af/A8/C fibre stimulation or the addition of 100 /SM-capsaicin. The elution time of authenticSP,-,, (@) and SP,-,, sulphoxide (0) are shown.

did not alter the assay (1000 ,UM) were without effect on either the resting or nerve-evoked release of SP-l.i. (Fig. 12). In contrast, the addition of the a2-agonist, ST-91,resulted in a reduction of the release of SP-l.i. and attenuated the increase in M.A.B.P.and pupil diameter caused by sciatic nerve stimulation (Fig. 13). This effect wasconcentration-dependent and occurred in the absence of any detectable effect onnon-stimulated release. These effects of ST-91 were antagonized by equimolar con-centrations of phentolamine. In two experiments a similar reversal was obtainedwith yohimbine (100 /lM) (data not shown).

Inhibitory amino acids. The addition of GABA (1000,M), baclofen (100,M) orglycine (100 ,UM) had no effect on the resting release of SP-l.i. in the spinal perfusates.

159


Muscimol (100 /M) produced a small but statistically significant reduction in therelease of SP-l.i. evoked by nerve stimulation (71 +6% ofcontrol stimulation). Higherconcentrations of baclofen or glycine were not examined. The addition of picrotoxin(100 /M) to the perfusate had no effect on the resting release but facilitated the releaseof SP-l.i. evoked by sciatic nerve stimulation (135+11 % of control stimulation).Picrotoxin enhanced the resting blood pressure (118+12 to 136+15 mmHg) andexaggerated the physiological response to nerve stimulation (mean increase in bloodpressure: 36+ 5 vs. 58 + 9 mmHg).

Characterization of SP-l.i.Fig. 14. presents the elution profile of SP-l.i. in pooled perfusates obtained during

resting conditions and the release evoked by sciatic nerve stimulation (30 x m.m.t.)or by capsaicin (0'1 mM). In all instances, SP-l.i. was found to be present as the majorpeak which co-elutes with authentic SP1 l. A smaller peak eluted immediately priorto the larger peak. This latter peak co-eluted with the oxidized form of SP; thisinterpretation is supported by the observation that the smaller peak disappearswhen the sample is pre-treated with the reducing agent mercaptoethanol; the entireSP,-,, peak will shift to the oxidized peak when treated with H202. Immunoreactivityevoked by sciatic nerve stimulation co-eluted with authentic SP on a G-50 column. Noother peaks were seen (data not shown).

Assays of serial dilutions (from 1: 1 to 1: 75) that were carried out with a pooledsample of the perfusate obtained during stimulation, yielded ligand displacementcurves which were parallel to those obtained with authentic SP.

DISCUSSION

Angelucci (1956) demonstrated that a material with SP-like properties was releasedfrom frog spinal cord by cutaneous stimulation. Early studies showing the presenceof SP-like material in spinal cord led to its nomination as a possible spinal neuro-transmitter (see McLennon, 1973 for early references). Physiological studies (Otsuka& Konishi, 1976; Urban & Randic, 1984) have demonstrated that SP meets thecriteria for an agent which mediates slow e.p.s.p.s in the spinal cord. The presentstudies indicate that high-threshold stimulation of the sciatic nerves will evoke astimulus-dependent increase in the level of ST-l.i. released from the spinal cord. Theco-elution of SP-l.i. obtained in the perfusate with authentic SP on two columnsystems along with parallel dilution curves confirm previous reports that the principalmaterial released into spinal extracellular space consists of the eleven-amino-acidpeptide (Akagi et al. 1980).The present experiments were designed to address the questions: (1) from whence

does the spinal SP-l.i. originate and (2) what neurotransmitter systems modulate itsrelease ?

Origins of spinal SP-l.i. release evoked by afferent stimulationAs discussed in the Introduction, immunohistochemical data point to the presence

of SP-l.i. in bulbospinal pathways and in type B ganglion cells in the spinal cord. Inthe present work, several lines of evidence support the hypothesis that the SP.l.i.

160


released from spinal cord by afferent stimulation derives from the activation of small,probably unmyelinated primary afferents.

First, although spinal 5-hydroxytryptamine is released by afferent stimulation(Tyce & Yaksh, 1981) and brain-stem stimulation releases spinal SP-l.i. in rats(Takano et al. 1984), neither cold block of the spinal cord nor treatment with 5,6-dihydroxytryptamine diminished the release of SP-l.i. evoked by afferent stimu-lation.

Secondly, the electrical threshold, associated with SP-l.i. release correlates withthe intensity of stimulation which evokes a component of the compound actionpotential, conducting at 1-2 m/s or less.

Thirdly, examination of the double-pulse experiments reveals that the apparentrefractory period of the system releasing SP-l.i. is greater than 10 ms, suggesting asmall-diameter fibre with a long refractory period. Although at this interpulseinterval a significant reduction occurs in the amount of SP-l.i. released and a smallafferent transmission failure occurs, it is not possible to distinguish between a failureof the release mechanism and an inability of the axons to follow the imposeddepolarization.

Fourthly, acute exposure of the spinal cord to the homovanillic acid derivative,capsaicin, results in a highly significant increase in the efflux of SP-l.i. from spinalcord in vivo (Jhamandas, Yaksh, Harty, Szolscanyi & Go, 1984; present studies) andsubsequent desensitization to further stimulation. Intracellular recordings haveshown that capsaicin-like compounds evoke a dose-dependent depolarization ofdorsal root ganglion cells which appears to be irreversible in small ganglion cells(Williams & Zieglgiinsberger, 1982). Electrophysiological classification suggests thatafferents activated by capsaicin can conduct at a C-fibre velocity and are sensitiveto thermal and chemical stimuli (e.g. bradykinin) (Fitzgerald & Wolf, 1982;Kaufman, Iwamoto, Longhurst & Mitchell, 1982). The absence of any detectabledecline in the levels of spinal SP-l.i. immediately after acute capsaicin treatmentindicates that the failure of sciatic nerve stimulation to evoke release was not due to ageneral depletion of SP-l.i. Several alternatives are possible. The amount of SP-l.i. ina readily releasable pool may be small in relation to the total store and may bedepleted by capsaicin. Alternatively, the releasible stores of SP-l.i. may be adequatebut capsaicin produces a long-lasting inactivation of the releasing terminals. Thelatter suggestion appears probable on the basis of electrophysiological data (Williams& Zieglgiinsberger, 1982). Since repeated stimulation at up to three discrete timesfailed to evoke any significant reduction in release (Fig. 3) it is probable that thereleasable stores of SP-l.i. remain adequate. Winter & Keen (1983) observed a smalldecline in rat cord levels of SP-l.i. after an extended interval of hours of stimulation,suggesting that it is possible to diminish releasible stores of the peptide.

Chronic pre-treatment with intrathecal capsaicin appears to result in a neurotoxiceffect largely limited to certain classes of peptide-containing unmyelinated primaryafferents (Jansco, Hokfelt, Lundberg, Kiraly, Halasz, Nilsson, Terenius, Rehfeld,Steinbusch, Verhofstad, Elde, Said & Brown, 1981; Nagy & Hunt, 1983). In thepresent studies intrathecal capsaicin had no effect on the levels of neurotensin andVIP in the dorsal and ventral horns. A 40-60% reduction was observed in SP-l.i.levels in the dorsal but not in the ventral horns. These residual stores are presumablyassociated with bulbospinal pathways and intrinsic neurones.

PHY 391

161


Taken together, this evidence supports the hypothesis that SP-l.i. released bysciatic nerve stimulation derives from terminals associated with afferents whichtappear to conduct at velocities corresponding to unmyelinated fibres. With regard tothermal stimuli (75°C) applied repetatively to the skin, a mild elevation (<40%) ofSP-l.i. release was found in five of five cats. In the cat, the modest increase evokedby thermal stimulation relative to electrical activation may be due to the near-maximum activation of the nerve terminals by the electrical stimulus. Interestingly,unlike with control or electrical stimulation experiments, a gradual rise in the baseline was noted after prolonged thermal stimulation (Fig. 2). The temperature of theprobe and its repeated application caused oedema and signs of inflammation. Thereis the possibility that the progressive rise might have been due to a developingcutaneous hypersensitivity secondary to the prolonged thermal stimulation. Theseresults are in accord with the recent report by Duggan & Hendry (1986), who useda novel antibody-coated micro-electrode and showed that increased levels of extra-cellular SP occurred in the substantia gelatinosa following activation of un-myelinated afferents. In addition, Duggan and colleagues have demonstrated spinalSP release due to high-intensity thermal stimulation (A. W. Duggan, personal com-munication).

Spinal systems which modulate primary-afferent-evoked SP-l.i. releaseIn the present studies, the administration of opioid agonists (morphine, sufentanil,

[D-Ala2-D-Leu5]enkephalin, and [D-Pen2-D-Pen5]enkephalin, and of an a2-adrenergicagonists (ST-91) resulted in a significant reduction in the release of spinal SP-l.i.evoked by small afferent stimulation. We believe these observations reflect themodulatory role of the respective receptor systems in spinal cord.

Several points should be considered. First, the minimum dose which producessignificant effects were in the range of 10-100 /M. It is probable that these highconcentrations reflect the steep diffusion gradients within tissue for both hydrophilicand hydrophobic agents (Herz & Teschemacher, 1971; Fenstermacher & Patlak,1975). Secondly, although the precise concentrations of these drugs in the tissue arenot known, it is reasonable to conclude that the tissue concentration is related to thatpresent at the spinal cord surface. Therefore it is important to note that, whereexamined, the effects were dose dependent. Thirdly, where a positive effect wasobserved, the nature of the receptor was assessed with the use of the respectivereceptor antagonist.

Opioid binding sites for [3H]dihydromorphine and [D-Ala2-D-Leu5]enkephalinhave been shown to occur in high concentrations in the dorsal grey and in dorsal root(Atweh & Kuhar, 1977; Fields, Emson, Leigh, Gilbert & Iversen, 1980) and todiminish in the dorsal grey after rhizotomy and capsaicin treatment (LaMotte, Pert& Snyder, 1976; Gamse, Holzer & Lembeck, 1979; Ninkovic, Hunt & Kelly, 1981;Ninkovic, Hunt & Gleave, 1982). Opioids alter the excitability of C-fibre primaryafferents (Sastry, 1980), and also alter ionic permeability in dorsal root ganglioncultures (Dunlap & Fischbach, 1981; Werz & MacDonald, 1983). These observationsaccount for the in vitro observations that ligands for ,u- and 8-receptors diminish theK+ release of SP-l.i. from medullary and spinal cord slices (Jessell & Iversen, 1977;Pang & Vasko, 1986), from dorsal root ganglion cell cultures (Mudge et al. 1979) and

162


that evoked by sciatic nerve stimulation in cat (Yaksh et al. 1980) and rabbit(Kuraishi, Hiroti, Sugimoto, Satoh & Takagi, 1983). The present studies indicatethat stimulation by ,s- and 6-ligands diminishes the levels of SP-l.i. in a naloxone-reversible fashion. Although not examined systematically, the effects of the agonistmorphine and the antagonist naloxone were stereospecific, since the activity isobtainable only with the (-)-isomers. Strict proof that the effects are mediated bytwo opioid receptors cannot be obtained from the present data, since the effects wereproduced by rather high concentrations of selective ,s-ligands (morphine and suf-entanil), and selective d-ligands [D-Ala2-D-Leu5]enkephalin and [D-Pen2-D-Pen5]-enkephalin) (Yaksh, 1984a, b). However, these observations are consistent with thein vitro physiological data and the ligand binding data where it- and 8-receptors havebeen identified in dorsal root ganglion. U50488H, a specific ligand for the K-receptor(Lahti, Von Voigtlander & Barsuhni, 1982), was without effect. Although K-receptorsare present in the spinal cord (Attali, Gouarderes, Audigier & Cros, 1981; Traynor,Kelly & Rance, 1982), our findings suggest that such receptors are not associatedwith effects on afferents from which SP is released.

Significant levels of binding for the 2-adrenergic antagonist [3H]rauwolscine andthe a1-adrenergic antagonist [3H]prazosin have been identified in the dorsal horn(Seybold, 1986). In the present paper it has been shown that ax2-ligands reduce therelease of SP-l.i. The ability to alter primary afferent excitability with a2-agonistsand antagonize the effects with yohimbine (Jeftinija, Semba & Randic, 1981;Calvillo, Ghignone & Quintin, 1985) is consistent with our observations. The presentresults are comparable to work reported by Kuraishi, Hirota, Sato, Kaneko, Satoh& Takagi (1985b) on the spinal release of SP-l.i. in the rabbit.Ample data have demonstrated that serotonin terminals (Ruda, Coffield & Stern-

busch, 1982) as well as [3H]5-hydroxytryptamine ligand binding sites are in highconcentrations in dorsal spinal grey matter (Seybold, 1986), but the present studyindicates that 5-hydroxytryptamine-sensitive sites are not relevant to the excit-ability of SP containing terminals.As far as inhibitory amino acids are concerned, we have found that they appear not

to be significantly involved in the modulation of the release of SP-l.i.In the present in vivo experiments in the cat, the pharmacological characteristics

of the release of SP-l.i. evoked by stimulation resemble the effects observed in brainslices. Thus, opioid ligands with ,s- or &-affinities have been shown to suppressdepolarization-evoked release in a naloxone-reversible fashion (Jessell & Iversen,1977; Pang & Vasko, 1986). Pang & Vasko (1985) have similarly reported a sup-pressant effect of noradrenaline but no effect of either 5-hydroxytryptamine orGABA. The latter result confirms the previous report of Sawynok, Kato, Navlicek &Labella (1982) who failed to observe any effect of baclofen on SP-l.i. release fromspinal slices.

SP as a primary-afferent transmitterJontophoretically applied SP has a potent excitatory effect on wide dynamic range

neurones in the dorsal horn (Henry, 1976; Murase & Randid, 1984). The slow excita-tory post-synaptic potential in spinal cord evoked by afferent stimulation ismimicked by the application ofSP and blocked by capsaicin. The electrophysiological

6-2

163

V. L. W. Go AND T. L. YAKSH

effects of afferent stimulation are blocked by analogues of SP with apparentantagonist activity (Urban & Randic, 1984). These findings, in conjunction with therelease of SP-l.i. from spinal cord by afferent stimulation, suggest that this peptideserves as a participant in the post-synaptic excitatory effects evoked by stimulationof certain classes of primary afferents. The fact that its release is evoked by thermaland mechanical stimuli (Duggan & Hendry, 1986; but see Kuraishi et al. 1985a),suggests that at least a population of the small primary afferents may be related toafferents that serve in the coding of activity evoked by high-intensity thermalstimulation. It is significant that the direct spinal activation of ,u- and 6-opioid anda2-adrenergic receptors, but not of adrenergic a, or opioid K receptors inhibit theresponse to a noxious thermal stimulus (Reddy, Maderdrut & Yaksh, 1980; Howe,Wang & Yaksh, 1983; Schmauss & Yaksh, 1984; Schmauss, Shimohigashi, Jensen,Rodbard & Yaksh, 1985; Yaksh, 1985). Although antinociception produced by theintrathecal administration of opioid agonists and a2-agonists, is unlikely to bemediated only presynaptically (Yaksh, 1985; Yaksh & Noueihed, 1985), these obser-vations provide correlary evidence consistent with the hypothesis that SP may playa role in the transmission from small high-threshold primary afferents. It is also clearthat SP is co-contained with other neuroactive agents in primary afferents and thatthe coding of the afferent message may be related to the characteristics of the afferentnerve pattern required for the differential release of SP (Dodd, Jahr, Hamilton,Heath, Matthew & Jessell, 1983; Urban & Randic, 1984).

We would like to particularly thank Ms Gail Harty for her unstinting and patient technicalassistance in the performance of these experiments. We would also like to thank Ms Ann Rocka-fellow for preparing the manuscript, Ms Kathleen Yaksh for graphics, and Ms Jane Bailey andMs Sharon Chinnow for performing the substance P and monoamine assays, respectively. Wewould like to acknowledge that the early phases of this work were initiated in collaboration withDrs Thomas Jessell, Susan Leeman and Ranier Gamse, and we thank them for their comments andsuggestions. This work was supported in part by NIH grants NS-16541 (T.L.Y.) and AM-34988(V.L.W.G.).

REFERENCES

ABAY III, E. 0. (1982). Capsaicin: production ofthermal analgesia and effects on substance P in thespinal cord in cats. M.Sc. Thesis, University of Minnesota.

AKAGI, H., OTSUKA, M. & YANAGISAWA, M. (1980). Identification by high-performance liquidchromatography of immunoreactive substance P released from isolated rat spinal cord. Neuro-8cience Letter8 20, 259-263.

ANGELUCCI, L. (1956). Experiments with perfused frog's spinal cord. British Journal of Pharmacol-ogy 11, 161-170.

ATTALI, B., GOUARDERES, Y., AUDIGIER, Y. & CROS, J. (1981). Multiple binding sites for 3H-ethylketocyclazocine on the lumbo-sacral portion of the guinea-pig spinal cord. In Advance8 inEndogenous and Exogenous Opioids, Proceedinga of the International Narcotic Research Conference,pp. 33-35. Tokyo: Kodansha Ltd.

ATWEH, S. A. & KUHAR, M. J. (1977). Autoradiographic localization of opiate receptors in ratbrain. I. Spinal cord and lower medulla. Brain Research 124, 53-67.

CALVILLO, O., GHIGNONE, M. & QUINTIN, L. (1985). Selective presynaptic depolarization of C fibreterminals in the feline spinal cord. Society for Neuroscience Abstracts 11, 410.

CUELLO, A. C., JESSELL, T. M., KANAZAWA, I. & IVERSEN, L. L. (1977). Substance P: localizationin synaptic vesicles in rat central nervous system. Journal of Neurochemi8try 29, 747-751.

164


DODD, J., JAHR, C. E., HAMILTON, P. N., HEATH, M. J. S., MATTHEW, W. D. & JESSELL, T. M.(1983). Cytochemical and physiological properties of sensory and dorsal horn neurons thattransmit cutaneous sensation. Cold Spring Harbor Symposium on Quantitative Biology 48, 685-695.

DUGGAN, A. W. & HENDRY, I. A. (1986). Laminar localization of the sites of release of immuno-reactive substance P in the dorsal horn with antibody coated microelectrodes. NeuroscienceLetters 68, 134-140.

DUNLAP, K. & FISCHBACH, G. D. (1981). Neurotransmitters decrease the calcium conductanceactivated by depolarization of embryonic chick sensory neurones. Journal of Physiology 317,519-535.

FENSTERMACHER, J. D. & PATLAK, C. S. (1975). The exchange of material between cerebrospinalfluid and brain. In Fluid Environment of the Brain, ed. CSERR, H. F., FENSTERMACHER, J. D. &FENCL, V., pp. 204-215. London, New York: Academic Press.

FIELDS, H. L., EMSON, P. C., LEIGH, B. K., GILBERT, R. F. T. & IVERSEN, L. L. (1980). Multipleopiate receptor sites on primary afferent fibres. Nature 284, 351-353.

FITZGERALD, M. (1983). Capsaicin and sensory neurones - review. Pain 15, 109-130.FITZGERALD, M. & WOLF, C. J. (1982). The time course and specificity of the changes in the

behavioral and dorsal horn cell responses to noxious stimuli following peripheral nerve capsaicintreatment in the rat. Neuroscience 7, 2051-2056.

GAMSE, R., HOLZER, P. & LEMBECK, F. (1979). Indirect evidence for presynaptic location of opiatereceptors in chemosensitive primary sensory neurones. Naunyn Schmiedebergs Archives ofPharmacology 308, 281-285.

GAMSE, R., LACKNER, D., GAMSE, G. & LEEMAN, S. E. (1981). Effect of capsaicin pretreatment oncapsaicin-evoked release of immunoreactive somatostatin and substance P from primary sensoryneurons. Naunyn Schmiedebergs Archives of Pharmacology 316, 38-41.

GREENWOOD, F. C., HUNTER, W. M. & GLOVER, J. S. (1963). The preparation of '31I-labelledhuman growth hormone of high specific radioactivity. Biochemical Journal, 89, 114-122.

HENRY, J. L. (1976). Effects of substance P on functionally identified units in cat spinal cord.Brain Research 114, 439-451.

HERZ, A. & TESCHEMACHER, H. (1971). Activities and sites of antinociceptive action of morphine-like analgesics and kinetics of distribution following intravenous, intracerebral and intra-ventricular application. Advances in Drug Research 6, 79-119.

HOKFELT, T., KELLERTH, J. O., NILSSON, G. & PERNOW, B. (1975). Experimental immuno-histochemical studies on the localization and distribution of substance P in cat primary sensoryneurons. Brain Research 100, 235-252.

HOKFELT, T., LJUNGDAHL, A., STEINBUSCH, H., VERHOFSTAD, A., NILSSON, G., BRODIN, E.,PERNOW, B. & GOLDSTEIN, M. (1978). Immunohistochemical evidence of substance P-likeimmunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervoussystem. Neuroscience 3, 517-538.

HOWE, J. R., WANG, J.-Y. & YAKSH, T. L. (1983). Selective antagonism of the antinociceptiveeffect of intrathecally applied a-adrenergic agonists by intrathecal prazosin and intrathecalyohimbine. Journal of Pharmacology and Experimental Therapeutics 224, 552-558.

HOWE, J. R., YAKSH, T. L. & TYcE, G. M. (1987). Intrathecal 6-hydroxydopamine or cervicalspinal hemisection reduces norepinephrine content, but not the density of a2-adrenoceptors, inthe cat lumbar spinal enlargement. Neuroscience (in the Press).

JANCSO, G., H6KFELT, T., LUNDBERG, J. M., KIRALY, E., HALASZ, N., NiLssON, G., TERENIUS, L.,REHFELD, J., STEINBUSCH, H., VERHOFSTAD, A., ELDE, R., SAID, S. & BROWN, M. (1981).Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide andmonoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin,neurotensin and 5-hydoxytryptamine. Journal of Neurocytology 10, 963-980.

JEFTINIJA, S., SEMBA, K. & RANDIC, M. (1981). Norepinephrine reduces excitability of singlecutaneous primary afferent C-fibers in the cat spinal cord. Brain Research 219, 456-463.

JESSELL, T. M. & IVERSEN, L. L. (1977). Opiate analgesics inhibit substance P release from rattrigeminal nucleus. Nature 268, 549-551.

JHAMANDAS, K., YAKSH, T. L., HARTY, G., SZOLCSANYI, J. & Go, V. L. W. (1984). Action ofintrathecal capsaicin and its structural analogues on the content and release of spinal substanceP: selectivity of action and relationship to analgesia. Brain Research 306, 215-225.

165


KAUFMAN, M. P., IWAMOTO, G. A., LONGHURST, J. C. & MITCHELL, J. H. (1982). Effect of capsaicinand bradykinin in afferent fibers with endings in skeletal muscle. Circulation Research 50, 133-139.

KuRAIsHI, Y., HIROTA, N., SATO, Y., HINO, Y., SATOH, M. & TAKAGI, H. (1985a). Evidence thatsubstance P and somatostatin transmit separate information related to pain in the spinal dorsalhorn. Brain Research 325, 294-298.

KuRAIsHI, Y., HIROTA, N., SATO, Y., KANEKO, S., SATOH, M. & TAKAGI, H. (1985b). Noradrenergicinhibition of the substance P-containing sensory primary afferents in the rabbit dorsal horn.Brain Research 359, 177-182.

KuRAISHI, Y., HIROTA, N., SUGIMOTO, M., SATOH, M. & TAKAGI, H. (1983). Effects of morphine onnoxious stimuli-induced release of substance P from rabbit dorsal horn in vivo. Life Sciences 33,693-696.

LAHTI, R. A., VON VOIGTLANDER, P. F. & BARSUHNI, C. (1982). Properties of a selective K agonist,U50488H. Life Sciences 31, 2257-2260.

LA MOTTE, C., PERT, C. B. & SNYDER, S. H. (1976). Opiate receptor binding in primate spinal cord:distribution and changes after dorsal root section. Brain Research 112, 407-412.

MCLENNON, H. (1973). Synaptic Transmission. Philadelphia: Saunders.MUDGE, A. W., LEEMAN, S. E. & FISCHBACH, G. D. (1979). Enkephalin inhibits release of substanceP from sensory neurons in culture and decreases action potential duration. Proceedings of theNational Academy of Sciences of the U.S.A. 76, 526-530.

MURASE, K. & RANDIC, M. (1984). Actions of substance P on rat spinal dorsal horn neurones.Journal of Physiology 346, 203-217.

NAGY, J. I. & HUNT, S. P. (1983). The termination of primary afferents within the rat dorsal horn:evidence for rearrangement following capsaicin treatment. Journal of Comparative Neurology218, 145-158.

NINKOVIC, M., HUNT, S. P. & GLEAVE, J. R. W. (1982). Localization of opiate and histamine H1-receptors in the primate sensory ganglia and spinal cord. Brain Research 241, 197-206.

NINKOVIC, M., HUNT, S. P. & KELLY, J.S. (1981). Effect of dorsal rhizotomy on the auto-radiographic distribution of opiate and neurotensin receptors and neurotensin-like immuno-reactivity within the rat spinal cord. Brain Research 230, 111-119.

OTSUKA, M. & KONISHI, S. (1976). Substance P and excitatory transmitter of primary sensoryneurons. Cold Spring Harbor Symposium on Quantitative Biology 40, 135-143.

PANG,I.-H. & VASKO, M. R. (1986). Morphine and norepinephrine but not 5-hydroxytryptamineand y-aminobutyric acid inhibit the potassium-stimulated release of substance P from rat spinalcord slices. Brain Research 376, 268-279.

REDDY, S. V. R., MADERDRUT, J. L. & YAKSH, T. L. (1980). Spinal cord pharmacology ofadrenergic agonist-mediated antinociception. Journal of Pharmacology and Experimental Thera-peutics 213, 525-533.

RUDA, M. A., COFFIELD, J. & STERNBUSCH, H. W. M. (1982). Immunohistochemical analysis ofserotonergic axons in lamina I and II of the lumbar spinal cord of the cat. Journal of Neuroscience2, 1660-1671.

SASTRY, B. R. (1980). Potentiation of presynaptic inhibition of nociceptive pathways as a mechan-ism for analgesia. Canadian Journal of Physiology and Pharmacology 58, 97-100.

SAWYNOK, J., KATO, N., NAVLICEK, V. & LABELLA, F.S. (1982). Lack of effect of baclofen on

substance P and somatostatin release from the spinal cord in vitro. Naunyn SchmiedebergsArchives of Pharmacology 319, 78-81.

SCHMAUSS, C. & YAKSH, T. L. (1984). In vivo studies on spinal opiate receptor systems mediatingantinociception. II. Pharmacological profiles suggesting a differential association of and Kreceptors with visceral chemical and cutaneous thermal stimuli in the rat. Journal ofPharmacology and Experimenta Therapeutics 228, 1-12.

SCHMAUSS, C., SHIMOHIGASHI, Y., JENSEN, T. S., RODBARD, D. & YAKSH, T. L. (1985). Studies on

spinal opiate receptor pharmacology.III. Analgetic effects of enkephalin dimers as measured bycutaneous thermal and visceral chemical evoked responses. Brain Research 337, 209-215.

SEYBOLD, V.S. (1986). Neurotransmitter receptor sites in the spinal cord. In FunctionalOrganization of Spinal Afferent Processing, ed. YAKSH, T. L., pp. 117-140. New York: PlenumPress.

166


TAKANO, Y., MARTIN, J. E., LEEMAN, S. E. & LOEWY, A. D. (1984). Substance P immunoreactivityreleased from rat spinal cord after kainic acid excitation of the ventral medulla oblongata: acorrelation with increases in blood pressure. Brain Research 291, 168-172.

TRAYNOR, J. R., KELLY, P. D. & RANCE, M. J. (1982). Multiple opiate binding sites in rat spinalcord. Life Sciences 31, 1377-1380.

TUCHSCHERER, M. M. & SEYBOLD, V. S. (1985). Immunohistochemical studies of substance P,cholecystokinin-octapeptide, and somatostatin in dorsal root ganglia of the rat. Neuroscience 14,593-605.

TYCE, G. M. & YAKSH, T. L. (1981). Monoamine release from cat spinal cord by somatic stimuli:an intrinsic modulatory system. Journal of Physiology 314, 513-529.

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

WERZ, M. A. & MACDONALD, R. L. (1983). Opioid peptides with differential affinity for mu anddelta receptors decrease sensory neuron calcium-dependent action potentials. Journal ofPharmacology and Experimental Therapeutics 227, 394-402.

WILLIAMS, J. T. & ZIEGLGiNSBERGER, W. (1982). The acute effects of capsaicin on rat primaryafferents and spinal neurons. Brain Research 253, 125-131.

WINER, B. J. (1962). Statistical Principles in Experimental Design. New York: McGraw-Hill.WINTER, E. & KEEN, P. (1983). Effects of synthesis inhibition and nervous activity on con-

centrations of neuronal substance P. Naunyn Schmiedebergs Archives of Pharmacology 323,173-175.

YAKSH, T. L. (1978). Analgetic actions of intrathecal opiates in cat and primate. Brain Research153, 205-210.

YAKSH, T. L. (1984a). Multiple opiate receptor systems in brain and spinal cord: part 1. EuropeanJournal of Anaesthesiology 1, 171-199.

YAKSH, T. L. (1984b). Multiple opiate receptor systems in brain and spinal cord: part 2. EuropeanJournal of Anaesthesiology 1, 201-243.

YAKSH, T. L. (1985). Pharmacology of spinal adrenergic systems which modulate spinal nociceptiveprocessing. Pharmacology, Biochemistry and Behavior 22, 845-858.

YAKSH, T. L. (1986). The central pharmacology of primary afferents with emphasis on the dis-position and role of primary afferent substance P. In Functional Organization of Spinal AfferentProcessing, ed. YAKSH, T. L., pp. 165-196. New York: Plenum Press.

YAKSH, T. L., ABAY, E. 0. & Go, V. L. W. (1982). Studies on the location and release of chole-cystokinin and vasoactive intestinal peptide in rat and cat spinal cord. Brain Research 242,279-290.

YAKSH, T. L., JESSELL, T. M., GAMSE, R., MUDGE, A. W. & LEEMAN, S. E. (1980). Intrathecalmorphine inhibits substance P release from mammalian spinal cord in vivo. Nature 286, 155-156.

YAKSH, T. L. & NOUEIHED, R. (1985). The physiology and pharmacology of spinal opiates. AnnualReview of Pharmacology and Toxicology 25, 433-462.

YAKSH, T. L., SCHMAUSS, C., MICEVYCH, P. E., ABAY, E. 0. & Go, V. L. W. (1982). Pharmacologicalstudies on the application, disposition and release of neurotensin in the spinal cord. Annals of theNew York Academy of Science 400, 228-243.

167

Documents

1 /M), and for the 8-receptor (D-Ala2-D-Leu6-enkephalin, 1-10 ,zM