Analgesic activities of spinal cord substance P antagonists implicate substance P as a neurotransmitter of pain sensation

Brain Research, 385 (1986) 74-85

74

Elsevier BRE 12068

Analgesic Activities of Spinal Cord Substance P Antagonists Implicate Substance P as a Neurotransmitter of Pain Sensation M.F. P1ERCEY, M.W. MOON, J.R. BLINN and P.J ,K. DOBRY-SCHREUR

CNS Research, The Uplohn Company. Kalamazoo, M149001 (U.S.A.) (Accepted 11 March 1986)

Key words: Analgesic - - Capsaicin - - Mouse - - Pain - - Scratch - - Spinal cord - - Substance P - - Substance P antagonist

Substance P (SP) injected into intraspinal 0.s.) spaces caused mice to vigorously scratch and bite their skins in an apparent reaction to a perceived cutaneous sensation. The scratching behavior was similar to the reciprocal hindlimb-scratching syndrome (RHS)described for intracranial (i.e.) SP injections. Radiotracer experiments, as well as potency and latency measurements, demonstrated that SP-induced scratching, whether induced by the i.c. or i.s, route, was due to SP receptor stimulation in the cervicothoracic cord. Similarly. biting was due to SP stimulation of the lumbosacral spinal cord. Mice coated with capsaicin, an irritant chemicalr scratched and bit the coated areas in a manner similar to animals injected with i.sl SP. Standard analgesics depressed this scratching behavior elicited by topical capsaicin. Non-analgesic drugs, with the exception of amphetamine, did not affect capsaicin-induced pain: It is concluded that i.s. SP induces a painful sensory experience. Some piperazinone derivatives of substance P's C-terminal hexapeptide are shown to specifically antagonize the scratching induced by i.s. SP with little or no effect on motor behavior. These antagonists depressed scratching elicited by topical capsaicin and were analgesic on the hot-plate test. It is concluded that SP is a natural neurotransmitter for pain and that antagonism of endogenous SP systems causes analgesia.

INTRODUCTION When the principal neurotransmitters for skeletal and visceral efferents were being elucidated more than 50 years ago, little was known about the identity of primary afferent neurotransmitters. In 1953, Lembeck ~l noted that the neuropeptide substance P (SP) was more concentrated in the dorsal roots than the ventral roots, and he suggested that SP might be a sensory neurotransmitter. Following elucidation of 8P's structure 6, immunohistochemical techniques demonstrated that SP is present in dense-core vesicles located in nerve terminals of small-diameter primary afferents making axodendritic contacts in the spinal cord dorsal horn :'7"8'12"34"53'55. Intense electrical stimulation of primary afferent fibers releases SP into spinal cord intrathecal spaces 71. Additionally, nociceptive n e u r o n s of the spinal cord dorsal horn are excited by SP 33"57"62, suggesting that SP is a neurotransmitter for primary nociceptive afferents. Positive identification of a substance as a neuro-

transmitter requires not only that the substance be present and released, but that the substance mimic the actions of the natural n e u r o t r a n s m i t t e r and that its antagomsts block the actions of the neurotransmitter 1768. In the present report, we demonstrate that when SP stimulates receptors in the spinal cord. mice behave in a m a n n e r similar to that displayed when their skins are coated with irritant chemicals. Additionally, we describe a novel group of selective SP antagonists which display analgesic activity. It is concluded that SP has fulfilled most of the major criteria traditionally required to accept a substance as a probable neurotransmitter. Preliminary accounts of some of this work have already been published 56"59-61 . MATERIALS AND METHODS

Behavioral experiments CF-1 mice (18-22 g, usually 6 mice per group) were used throughout. Intracranial (i,c.) injections

Correspondence: M.F. Piercey, CNS Research, The Upjohn Company, Kalamazoo, MI 49001. U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical DivisionJ

75 were made with drug in 2/~1 of 0.9% saline at a depth of 3.5 mm almost at the midline and just anterior to the level of the external auditory meatus. When such injections were made with dye-filled syringes, the dye was observed to fill the third ventricle, the two lateral ventricles and occasionally the fourth ventricle. Intraspinal (i.s.) injections were made between the most caudal lumbar vertebrae, successful insertion being confirmed by a short tail-flick upon penetration. In almost every case, such penetration elicited no other overt effects which would be suggestive of damage. However, on very rare occasions, hindlimb paralysis occurred. Animals with such hindlimb paralysis were immediately rejected. As with i.c. injections, i.s. injections were 2 M. When 0.1% cresyl violet was injected i.s., the dye was located in the intrathecal spaces surrounding the lumbar cord, but not in the more cephalad cord. Later experiments using radiotracer demonstrated that actual drug distributions were more widespread than indicated by the dye experiments (see below). Following injections, animals were placed into individual plastic chambers with fine wood shavings on the floor. Animal behaviors were observed for 5 min and scored quantally for the presence or absence of the scratching syndrome previously described 14. Potencies were calculated as EDs0's from their d o s e response curves according to the S p e a r m a n - K a r b e r method (doses were 0.5 log intervals apart, 6 animals being tested with each dose). Animals injected with somatostatin (SRIF) were scored for one-sided scratching, whereas animals injected with SP were scored for reciprocal hindlimb scratching (RHS, see ref. 14). Latencies and the amount of time spent doing each behavior were measured by stopwatches with 3 observers doing the measurements on each mouse (one for hindlimb scratching, one for biting and one for facewipe "grooming-type' behavior). Antagonists were usually co-administered with 74 pmol of SP. Antagonism was judged as complete absence of the biting and scratching syndrome over a 5minute observation period. Doses of antagonists were separated by half-log intervals, with 6 animals being tested at each dose. Potencies were measured as the dose causing complete SP antagonism in 50% of the animals, according to the S p e a r m a n - K a r b e r method > . To control for non-specific effects, active antagonists were tested for their ability to inhibit

SRIF scratching. Chemical irritants were applied to cutaneous surfaces by means of a Q-Tip soaked in an appropriate solution. The duration of application and size of area coated were rigorously kept as identical as possible for all animals, whether they were treated with irritant or vehicle control. Ears were coated over a 30-s period, care being taken to insure that both sides of the ear were completely coated without touching subjacent cutaneous areas. Feet were coated over a 20-s period, and shaven back areas were coated by a single dab of the Q-Tip. Animals were observed closely for scratching and biting, which were scored as the number of events during the observation period of 5 (or, during antagonist testing, 3) rain immediately following irritant application. SP antagonists were injected 30 s prior to irritant application to shaven backs. Control groups were run in parallel with treated groups. The dose required to reduce the number of events in a 3-rain observation period by 50% was defined as the E D > . This E D > was measured by interpolation of the dose-response curves. The tail-flick test was adapted from D ' A m o u r and Smith II. The distance and intensity of a focused lamp were adjusted to give control latencies of 3 - 4 s to flick. The hot-plate test ~) used a hollow copper plate enclosed by a 23 cm diameter inward-sloping glass wall 11 cm high. The plate was filled with flowing water, heated to attain a uniform surface temperature of 55 °C. The time to paw lick (invariably the forepaws) was measured; control latencies were 6 - 7 s and 20 s was arbitrarily selected as a cutoff. In experiments where SP itself was tested for analgesic activity, the time to hindpaw lick was also measured. In these experiments, cutoff time was 30 s. In all of the thermal analgesic tests, an animal was judged to be analgesic if the latency to respond exceeded the control values by more than two standard deviations.

Radiotracer experiments SP solutions spiked with radiolabeled Tvr s SP (3 uCi ~25I/mol) were injected by each route. As soon as the scratching syndromes were unambiguously initiated, animals were instantly frozen by immersion in liquid nitrogen to prevent further diffusion. To prevent diffusion or degradation, animals were dissected in the frozen state by transections at the cervicalcranial and thoracic-lumbar junctions: spinal col-

76 umns were further segregated from the rest of the body. The distribution of radioactivity was then measured by solid scintillation counting. Chemical methods The modified hexapeptides were prepared in solution by standard peptide-coupling procedures, using dimethylformamide as the solvent and dicyclohexylcarbodimide-hydroxybenzotriazole as the coupling reagent. Peptide products were purified at each reaction step by chromatography on silica gel using chloroform-methanol as eluent. The modified dipeptide fragments 3(R)-((t-butoxy)carbonyl)amino-(S)-(2methylpropyi)-2-oxo-l-pyrrolidineacetic acid and the (S),3(R) and (S),3(S) isomers of 4-((t-butoxy)carbonyl)-2-oxo-a,3-bis(phenylmethyl)-l-piperazineacetic acid were prepared using modifications of literature procedures24'4~; all other protected amino acids required in this work were obtained commercially. RESULTS Scratching induced by i.s. SP When 22 pmol of SP were injected intraspinally, the mice began to bite their hindlimbs, abdomen and groin in a very few seconds (Table I). The biting was directed alternately to the left and right, in a manner reminiscent of the reciprocal hindlimb scratching (RHS) syndrome observed following larger SP doses given intracranially 14. Approximately 20 s following the i.s. injection, the mice would exhibit R H S virtually indistinguishable from that following i.c. SP (except that the behavior was interrupted by foot-biting episodes). The R H S was directed mostly to the thoracic regions. Approximately 1.5 min later, the animals would lick their forepaws and wipe their facial

skin (Table I). All of these behaviors appeared to reflect responses to perceived sensory experiences. The animals precisely directed their paws and mouths to cutaneous surfaces: the behaviors appeared to be highly purposeful. In every animal, the sequence was always the same: hindpaw biting preceded scratching, which preceded forelimb biting (Table I). Animals injected i.s. spent a much greater time licking and biting their hindquarters than did animals injected i.e. (Fig. 1A). SP was considerably more potent in eliciting scratching/biting by the i.s. route (EDs0 = 1.3 pmol, 959~ C.I. -:: 0.76-2.2 pmol) than by the i.e. route (EDs, = 22 pmol, 95~::,~('.l. =15.4-31.4 pmol). When pharmacologically equivalent doses were tested, RHS had an earlier onset following i.s. injections (12 _+ 5 s, mean _+ S . E . M . ) t h a n i.e. injections (57 _+ 19 s). RHS was not mimicked or antagonized by i.p., s.c. or i.v. SP administration up to 5(/mg/kg. Also, whether administered i.s. or i.e., no RHS could be obtained from acetylcholine, adrenocorticotrophic hormone ( A C T H ) , ACTH4_~il, atropine, bradykinin, D O P A , ethanol, gastrin, glutamic acid. histamine, morphine, neurotensin, norepi-

A

BEHAVIORAL EFFECTS

INJECTION

220 pMOLESSP

TABLE I Latencies for behavioral effects in mice following intraspinal inkction of substance P Values are the means +_ S.E.M. in s following 22 pmol SP i.s. (n = 6). Hindpaw biting includes biting hindpaw, tail, scrotum. abdomen or adjacent cutaneous surfaces. Forepaw wiping includes biting forepaws or wiping with forepaws. Hindpaw biting

Scratching (RHS)

Forepaw wiping

11.5 _+4.9

19.3 + 5.5

104.2 + 25.1

INJECTION 15 pMOLESSP

B

INTRASPINAL SP DISTRIBUTION

)t,IJECTIO~$ 220 pMOLES SP

iNJECTIONS 15 pMO[ESSP

Fig. 1. l'he relative amount of time spent back-scratching as compared to foot-biting is proportional to the relative amounts of SP delivered to the anterior as compared to the posterior spinal cord. A: the percent of the 5-rain observation time spent back scratching (solid columns) and foot-biting (dotted Columns) following pharmacologically equivalent doses of SP delivered i.c. (left) and i.s. (right). B: the distribution of SP tO the anterior (thoracolumbar) and posterior (lumbosaeral) spinal cord following injections of pharmacologically equivarent SP doses by i.c. (left) and i.s. (right) routes. Distribution determined by measuring tracer amounts of radi01abeled SP added to SP solutions.

77 nephrine, serotonin, thyrotropin-releasing hormone (TRH) or vasoactive intestinal polypeptide (VIP). Kainic acid and SRIF, however, did elicit relatively weak one-sided scratching syndromes. SP also elicited one-sided scratching with doses below those capable of producing RHS, suggesting that RHS involves a more intense sensory perception than the single-sided scratching. Systemic capsaicin can cause analgesia by depletion of SP and other primary afferent neural mediators, presumably by damaging primary afferent unmyelinated fibers 52~:73~-3466"7°. In our hands, a single s.c. injection of 30 mg/kg capsaicin 24 h earlier made mice virtually insensitive to natural painful stimuli. All such mice were analgesic on the tail-flick test and, unlike untreated mice, did not wipe eyes topically treated with the irritant zingerone. However, mice treated with 30 mg/kg capsaicin 48 h earlier and demonstrated to be analgesic on the tail-flick and zingerone tests still scratched vigorously to a 100 ng i.s. injection of SP. Similarly, sectioning the sciatic nerve did not stop the mice from biting their ipsilateral paws in response to i.s. SP. Since the primary afferent nociceptive fibers were not functional, SP must have elicited the behavioral response by direct stimulation of secondary afferent neurons located within the cord. When radiolabeled SP was injected, i.s. injections were distributed principally to spinal regions, with negligible amounts found in the head. When radiolabeled SP was injected i.c., SP was recovered principally from the head, with less material found in the anterior spinal column: negligible amounts were located in the lumbosacral columns, with about half of the animals having no detectable levels in this area. However, when giving equivalent pharmacological doses, the absolute amounts of SP recovered from the rostral spinal columns following i.c. injections were actually slightly greater than that recovered from animals given i.s. injections (Fig. 1B). Since the cervicothoracic column was the only area that was reliably reached by both i.s. and i.c. routes, this area appears responsible for eliciting the sensations underlying the scratching syndrome common to both routes. The relative amount of biting as compared to scratching was similar to the relative distribution to the lumbosacral as compared to the cervicothoracic column (Fig. 1).

A EAR-SCRATCHING

B BACK-SCRATCHING

T

25

¢:= C:3 rJ 3 B

.,=,

P, ..=.

Z

CAP

VEHICLE

CAP

VEHICLE

Fig. 2. Number of scratching episodes (mean _+S.E.M., n = 8) on capsaicin-coated skin (solid bars) as compared to vehiclecoated skin (dotted bars). A: capsaicin :and vehicle are applied to ears with a 5-min observation time beginning immediately after application with a Q-Tip. B: as in A except that capsaicin and vehicle are applied to discrete areas of shaven backs. Asterisks in A and B indicate that number of episodes on capsaicin-coated skin are significantly different from the number of episodes observed on the vehicle-coated skin (P < 0.05, Student's t-test).

Scratching induced by cutaneous irritants Capsaicin, the active ingredient of Hungarian red peppers, produces intense pain when applied to skin 47. When 1% alcoholic solutions of capsaicin were applied to the ear of a mouse, the ear would rapidly (within 60 s) begin to inflame intensely. The opposite ear, coated with vehicle, did not inflame. The mice scratched their inflamed ears frequently, but only rarely and weakly wiped the vehicle-coated ears (Fig. 2A). This scratching, using either the forelimb or the hindlimb, was most intense during the first 5 rain following onset of inflammation, but persisted throughout a 20-rain observation period. When 1% alcoholic capsaicin solutions were applied to the hindpaws, the animals bit these paws quite intensely, but never bit the opposite feet coated with vehicle (Fig. 3). This behavior was most intense during the first 5 min of the observation period (which followed a 2-min waiting period to allow inflammation to set in). Once biting began, some capsaicin would rub onto the animals' faces, which also became

78 A

50

BITING EPISODES

B

50

•

BITING TIME

Effects of standard drugs on scratching induced by toptcat capsaicin

T

Values are the intraperitoneal doses required to reduce thc number of scratching episodes bv 5()f/cin the 3-rain period following capsaicin application Drugs were rejected at maximal doses not causing overt behavioral effects I.A.. reactive

5=

=E

g

0

41CLE

0

TABLE II

[)rug clas,s

Drug

ED~o (true/k~,,

Analgesic Analgesic An algeslc Antipsychotic Antidepressant Anxiolvtic Muscle relaxant Stimulant

Morphine Aspirin Nalorphine Chlorpromazme lmipramine Diazepam Baclofen Amphetamine

3 100 1() 1.A I. A 1.A I. A

!Fli~;LE

Fig. 3. Intensity of hindquarter biting activity after applying capsaicin (solid bars) and vehicle (open bars, all values zero) to feet by Q-Tips. A: number of biting episodes (mean _+ S.E.M., n = 8) in 5-min observation time which was begun 20 s after application. B: amount of biting time in s (mean + S.E.M., n = 8) in 5-min observation time which was begun 20 s after application. Asterisks in A and B indicate that values observed for capsaicin-coated skin are significantly different from the values observed for vehicle-coated skin (P < 0.05, Student's t-test).

some known analgesic effects > . did depress capsa> cin-induced scratching. Although the behaviors were slightly less intense. similar scratching/biting s y n d r o m e s were evoked bv cutaneous application o f m u s t a r d oil. another inflammatory irritant S P antagonism

inflamed. Thereafter, biting decreased (presumably because of punishment since the inflammation persisted). The 1% alcoholic capsaicin solutions did not produce inflammation when applied to shaven backs. Presumably, the alcoholic solutions were not penetrating the tougher hide covering the backs. However, when Oieoresin Capsicum (Kalsec Inc.) was applied to shaven backs, animals vigorously scratched this area (Fig. 2B). W h e n this capsaicin p r e p a r a t i o n was applied bilaterally to their shaven backs, they would exhibit classical R H S . This behavior was most intense in the first 5 min following irritant application. As capsaicin transferred to their hindpaws, they would begin to bite their hindpaws and, as it then transferred to their faces, they would decrease the biting and scratching, again p r e s u m a b l y because of punishment. Analgesic drugs such as m o r p h i n e , aspirin and naIorphine depressed capsaicin-induced scratching (Table II). Non-analgesics generally had little effect at doses below those eliciting overt behavioral effects. H o w e v e r , a m p h e t a m i n e , a stimulant with

Table I l l illustrates the structures and potencies of some piperazinone SP derivatives found to antagoraze scratching induced by i.s. SP. This effect was dose-related. Doses of 10-30/~g totally obliterated the scratching syndrome elicited by i,s. SP. The antagonism was specific to SP because these compounds did not antagonize the w e a k e r one-sided scratching s y n d r o m e elicited bv SR1F. Unlike DPro2-D-Phe7-D-Trp ~ SP discussed in our preliminary reports 5~'~). these piperazinone analogs caused little or no hindlimb paralysis. The piperazinone antagorests, like D-ProZ-D-PheV-D-Trp') SP. were inactive when injected i.v. even with doses as high as 30 mg/kg. On the rat colon. U-66,739 was a weak agontst. suggesting that the piperazinone antagonists may be more selective than the D-Phe and D-Trp analogs. which a p p e a r to be effective antagonists on a variety of tissues excited by SP 22'2359'~°68 Effects o f S P on analgesic tests It has been r e p o r t e d that SP itself, g~ven either systemically5t,67. intracranially 234<~v. or intra-

spinally 1s'3~. is analgesic. There are some inconsls-

79 T A B L E III

Analgesic ef/`ects of substance P antagonists following intraspinal injections Values are the doses in u g required to eliminate responses in 5(}% of animals (SP scratch, SRIF scratch, tail flick, hot plate) or reduce number of episodes by 51}% (capsaicin scratch) following i.s. injections. I.A., inactive at 3{)itg. N.T.. not tested.

Antagonism EDso'S SRIF scratch

Capsaicin scratch

Tail .~'lick

Hot plate

I.A.

4.8

I.A.

31)

1.A.

0.4

I.A.

I.A.

3.2

I.A.

2.7

I.A.

3[)

0.5

I.A.

0.3-3.0*

I.A.

SP

scratch

0 U -62.713

~-- -C--N

N--C- C- Gly- Leu -- Met - NH 2

1.6

5 -C--N

U-65,822

N D - C - C - D - P r o - L e u - M e t - NH 2

10

'o' >Jo ""

~o

©

© O~H

E

/---X

XF_C_ N

U-65,823

/

/o

II

CH 2 ND,- C- ,C,- D-- P r o - Leu - M e t - NH2

\

/

;

h

#---~o ~o

5 U-66.739

O l

/ /

HN~

H "x~_C_ N

II \

6 ~ o CH2

0

Nm,-C-C-NH'y

/

I ,

¢' o

"C-C-Met-NH 2

o

cH21 CH-CH 3

CH 3

'

8.3

80

(Table11lcontinued) 0

~.~

H

U-67,202

~ -

-

D.,.

-NH"

H ""

Met.-

NH 2

* Dose-response too flat to get a precise EDs 0, tencies between the results of different laboratories. Oddly, the effects do not occur within the first few minutes following injection when SP is eliciting dramatic behavioral actions. Rather, they occur 30, 6(1 or 90 rain later! In contrast to many of these studies, the report of Frederickson et al. 23 gives an adequate description of their methodology in order to attempt a replication. In that study, the investigators utilized the mouse hot-plate assay, relying solely on the response time required for hindpaw lick rather than on the more traditional time to forepaw lick. We have tried to replicate and indeed expand on that study.

,~ U-62,713

B U-65,822

We have found the time to hindpaw lick a much more variable response than the time to forepaw lick. Using a wide variety of doses (1.25, 2.5, 5 and 50 ng, and 1.25, 2, 5 and 50 ttg for central injections, 1-50 mg/kg for i.p. injections) latencies (10, 30; 60, 90 min), and injection routes (i.p., ilc., i.s:), we found no effects on the tail,flick or the traditional hot-plate test using time to forepaw lick. Only one i.c. dose at 60 min (but not 10, 30 or 90 min:) weakly increased hot-plate tatencies using the more variable time to hindpaw lick. Based on the number of tests per, formed, this is less of an effect than would be expected by chance alone. The data illustrated in the report of Frederickson et al: also indicate an extremely small effect for SP. We conclude that SP has little or no analgesic effect.

t0

100

Effects of SP antagonists on analgesic tests

C u-65,823

D u-66,739

~o =..

Fig. 4. Use of intraspinal injections of SP antagonists to depress scratching elicited by topical applications of capsaicin to shaven

backs of mice. Heights of bars (solid for vehicle-injected controls, dotted for SP antagonists) indicate the mean ,- S.E.M. number of episodes expressed as percent of vehicle-injected controls. Asterisks indicate that values obtained in SP antagonist-injected animals are significantly different from those m vehicle-injected animals (Student's t-test. *P < 0.05 or **P < 0.01). For each antagonist, 30/~g of peptide was intraspinally injected into each animal.

lntraspinal injections of the SP antagonists successfully suppressed scratching induced by topical capsaicm treatment (Table III, Fig. 4) just as systemically-administered analgesics do (Table IlL lntraspinal injections of the more potent SP antagonists were also effective as analgesics on the hot-plate test (Table Ill), an assay generally only sensitive to strong analgesics ~9. The most potent of the SP antagonists had potencies on the hot plate similar to those for the narcotic morphine when all were iniected intraspinally (Fig. 5). None of the SP antagonists were effective as analgesics on the capsaicin test or on the hot plate when injected intravenously at a dose of 30 mg/kg. In contrast, morphine depressed cap~icin scratching with an EDs0 of 3 mg/kg (Table II) and had an EDs0 of 3.8 mg/kg on the hot plate when injected intraperitoneally. Although some SP antagonists were active on the hot plate, none were effective in

81 With regard to localization, SP has been found within small-diameter primary afferents similar to those thought to be involved in sensory transmission of pain 34. Terminals of primary afferents have been found in the spinal cord dorsal horn containing SP-laden dense core vesicles 27"12'54'55. These terminals

20'-2.

(n

E

..I

+I

" E W

c

Controls

O--O

U-66,739

A-----~ M O R P H I N E

0.3

1'

3~ 1 Jo 3o D O S E (/~g i.s.)

1 0' 0

Fig. 5. Dose-response of SP antagonists, U-67,202 (solid circles) and U-66,739 (open circles), and morphine (triangles) on

the hot-plate test in the mouse. Abscissa gives i.s. doses tug, vehicle-controls to the left) on a logarithmic scale while ordinate gives time to paw lick (mean _+ S.E.M., n = 6) after treated animal was placed on a 53.5 °C hot plate.

antagonizing the flexion reflex as measured by prolongation of the time required to flick a tail out of the heat stimulus of a focused lamp beam (Table Ill). DISCUSSION

Based on classical descriptions 17, a neurotransmitter can be defined as a substance which when released from a nerve terminal by action potentials transmits by itself meaningful information, either excitatory or inhibitory, to a neuron or group of neurons lying in close apposition to the nerve terminal releasing the substance. Although SP has been demonstrated to excite dorsal horn nociceptive neurons, it has been suggested that SP cannot be a neurotransmitter because it acts too slowly to transmit meaningful information; rather, it has been suggested that SP is some sort of a neuromodulator 3339"4°62. In order to facilitate consensus, criteria for neurotransmitter identification have been proposed 17"69. The most critical include: (1) localization to the appropriate nerve terminals; (2) nerve impulse controlled release from these terminals; (3) mimicry of the natural transmitter's actions (physiological identity); and (4) susceptibility to pharmacological manipulations that affect the natural mediator (pharmacological identity).

make axodendritic contacts, confirming that SP is appropriately located to function as a synaptic transmitter. SP has also fulfilled the release criterion. Stimulation of low-threshold afferents, most likely involved in transmitting non-noxious sensory information, does not evoke release of SP. However, electrical stimulation of high-threshold afferents, similar to those thought to be involved in pain sensation, evokes the release of SP into spinal cord intrathecal spaces 71. The chemical irritant capsaicin also causes the release of SP 27-2')7~)-72. Substance P has also satisfied the criterion of physiological identity. SP excites dorsal horn neurons excited by noxious stimuli, but not those selectively excited by non-noxious stimuli 335s. The bilateral scratching and biting syndrome which we (and others, see e.g. ref. 35) have observed represents a dramatic demonstration of the identity of SP to a natural sensory transmitter. Although SP was administered centrally, the animals' responses were precisely directed toward their skin. Since a similar behavior is induced by coating the skin with algesic chemicals, the behavior is appropriate for an animal responding to a painful sensation. Other laboratories have also implicated scratching and biting as a typical rodent response to chronic or inflammatory pain 13~. The radiotracer experiments (Fig. 1) substantiate that the sensory experience was evoked bv SP at the spinal level because pharmacologically equivalent i.c. and i.s. doses delivered adequate amounts of SP to this site. Further evaluation of these data allows one to associate scratching with cervicothoracic SP receptors, and biting with stimulation of lumbosacral SP receptors. Foot-biting was rarely observed following i.c. injections, which delivered negligible peptide to the lumbosacral cord. In contrast, biting was the most dominant behavior following i.s. injections. which also delivered more SP to the lumbosacral cord than anywhere else. The observation that foot-biting always preceded back scratching following i.s. injections (Table 1) is also consistent with the notion that foot-biting is induced close to the lumbosacral injec-

82 tion site, but that scratching requires peptide diffusion to more rostral areas. The association of scratching and hindpaw-biting responses with cervicothoracic and lumbosacral cord, respectively, further substantiates the sensory nature of the SP stimulus. In the first case, hindlimb scratching of thoracic areas is being induced through the cervicothoracic region known to contain the appropriate sensory dermatomes, but not the motor area for hindlimb motion. In the second case, the lumbosacral SP distribution is appropriate for sensation in hindlimb skin, but not for the motor aspects involved in biting the hindlimb skin. This rules out any role for the presumably non-sensory crude scratching-like responses sometimes observed in spinalized animals t3. All of these data demonstrate that a primary afferent SP system exists which is capable of communicating meaningful sensory messages. The analgesic effects of the SP antagonists demonstrate that this primary afferent system is actually required for relaying information about at least some aspects of painful stimulation. Unlike the D-Trp and D-Phe derivatives discussed in our preliminary reports 59'6°, the piperazinone analogs described here lack paralytic effects. Because of this, we were able to test somewhat higher doses, which revealed that SP antagonists can antagonize responses to noxious heat as perceived on the hot-plate assay. As was true with the D-Trp and D-Phe derivatives, the piperazinone derivatives successfully blocked sensation associated with cutaneous irritants. Moreover, the painful nature of that sensation has now been demonstrated by noting the selective antagonism of these scratching responses by analgesic drugs. Antagonism of the natural response to pain stimulation by selective SP antagonists satisfies the final major criterion-pharmacological identity-for identification of a neurotransmitter substance. Moreover, the fact that the antagonists were effective only when delivered directly into the spinal column corresponds precisely with the proposed primary afferent role for SP. Previously, it has been shown that depletion of primary afferent SP by capsaicin treatment also causes analgesia 5'26'32"7°. Additionally, genetically analgesic patients lack primary afferent S P 49'53. However, other primary afferent transmitters are also absent in capsaicin-treated animals 28'66 and ge-

netically analgesic patients. The depression of SP release from primary afferents bv narcotic analgesics3771 is also consistent with the pharmacological identity of SP and a natural neurotransmttter for pain. However. narcotics also depress nociceptive dorsal horn neurons by postsynaptic mechanisms not i n v o l v i n g SP 16-58"73.

Although the SP antagonists were analgesic on the hot-plate and capsaicin assays, they were inactwe on the tail-flick assay. Since the hot-plate and capsaicin tests employ stimuli somewhat more diffuse than the highly focused lamp beam of the the tail-flick test. it is possible that SP is a neurotransmitter for intense diffuse pain but not for extremely sharp pain. Neurophysiological experiments 4 indicate that multiple pathways exist for pam transmission. The experiments reported here suggest that an agent other than SP may serve as the neurotransmitter for sharply defined pain. In summary, SP fulfills all the major criteria-localization, release, physiological and pharmacological identity-to qualify as a neurotransmitter for primary noeiceptor afferents. In contrast, the suggestion that primary afferent SP is a neuromodulator 33394°62 does not seem to be supported by the evidence. Although there is no precise definition of a neuromodulator, one concept is that of a mediator which, although it does not itself excite or inhibit neurons, modifies the ability of a neurotrarksm~tter to excite or inhibit neurons. In the dorsal h o r n SP can directly excite neurons by itselj53"58"62: t h u s the peptide clearly does not act as a silent facilitator or modulator of another specific neurotransmitter. An alternative definition for a modulator is a substance released into a more general environment than a synaptic cleft Le.g. something more akin to 'paracrme" secretion 251, which then facilitates or depresses the level of excitability of a general population of neurons in this larger region. In the dorsal horn, SP is located in nerve terminals making close synaptic connections with dendritic shaftsTA2'56: such a relationship is totally inconsistent with release into a more general environment. Moreover, SP excites only a minority of dorsal horn neurons, the effects being confined to nociceptive cells. It is commonplace for SP-insensitive cells to be located adjacent to SP-sensitive ones 3357ss~2 A general modulator of regional neuronal excitability would not be expected to show such strong speci-

83 ficity of effect. F u r t h e r m o r e , we have now shown that SP by itself transmits (rather than modulates) direct sensory information about the condition of an animal's skin. With regard to the slow time-course following iontophoretic SP, it cannot be expected that a substance administered exogenously at a distance of 300/~m or more away would act as rapidly as the same substance endogenously released into the synaptic cleft only 200 A or so away from the postsynaptic SP receptor. In fact, the time-courses for the excitation of dorsal horn neurons by iontophoresing SP 31y'Ss~2 parallel quite closely to those expected for a substance diffusing from a point source 100-300 u m away from its receptor~3~': such a distance is consistent with the dendritic location of SP synapses. Even though some evidence has now been garnered to support each major criterion for neurotransmitter identification, more work needs to be done. For example, because of difficulty in iontophoresing these peptides, SP antagonism at the level of the single dorsal horn cell has not yet been demonstrated. Additional desirable neurophysiological measures may be even more difficult to demonstrate. It is difficult, for example, to show that SP-sensitive nociceprive dorsal horn neurons are monosynaptically excited by high-threshold primary afferents because: (1) volleys of high-threshold afferents are too asynchronous and slow to get a precise measure of synaptic latency; (2) the dendritic location of primary afferents containing SP precludes m e a s u r e m e n t of conductance changes and precise times of EPSP initiation; and (3) many SP-sensitive dorsal horn neurons have convergent inputs from different types of primary afferents. However, it seems likely that SPas selective excitation of nociceptive dorsal horn neurons 3~'5s is related to primary afferent synapses since most dorsal horn SP is derived from this source 2 s 34.38. it is interesting to compare what is known about the role of SP in primary afferents to what is known

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about acetylcholine, which is widely accepted as the neurotransmitter utilized by m o t o n e u r o n recurrent collaterals to excite Renshaw cells 17. The monosynaptic connections between m o t o n e u r o n recurrent collaterals and Renshaw cells have been well demonstrated physiologically1s65, but have never been d e m o n s t r a t e d anatomically. In contrast, synapses between substance P-containing primary afferents and dorsal horn neurons have been well demonstrated anatomically 2"712"56, but have never been d e m o n s t r a t e d physiologically. For neither transmitter has it yet been d e m o n s t r a t e d that the effects on m e m b r a n e conductance are identical to those of the natural transmitter, although W e r m a n 69 suggested that when technically feasible this would be an ideal p a r a m e t e r for establishing physiological identity. We have d e m o n s t r a t e d a behavioral significance of primary afferent SP, but the behavioral role played by acetylcholine at the Renshaw cell has never been directly d e m o n s t r a t e d . The identification of SP as a p r o b a b l e spinal cord neurotransmitter for nociceptor afferents has been paralleled by experiments establishing its role as the mediator of the 'axon reflex '43 at peripheral nociceptor terminals 4245'64. Taken together the data support Dale's 1~ hypothesis, made 50 years ago, that the central and peripheral mediators of this system are identical.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of L . A . Schroeder in these experiments. P. K e m p assisted us with statistical evaluation of the data. Dr. V.H. Sethy and D . W . Harris graciously p r e p a r e d the [125I]Tyr-8 SP and Kalsec (Kalamazoo, MI) kindly provided the Oleoresin Capsicum and mustard oil,

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