Evidence that raphe-spinal neurons mediate opiate and midbrain stimulation-produced analgesias

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SUMMARY

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334 (eadorphins). tn a variety of mammalian species including r~an, behavioral analgesia can be produced by stimulation of many subcortical regions [20, 43] including hypothalamus [4,13,26], striatum [19,39,59] and periaquecontain significant concentrations of opia~ rec, endorphins [16,32,50,60,61]. One of the most thoroughly studied of these sites is the mesencephalic periaqueductal gray (PAG). Microinjection of opiate agonists into PAG produces powerful analgesia [ 11,35,38] and injection of opiate antagonists into this region blocks the action of systemically administered opiate agonists [35,64,65]. These observations indicate that the analgesia produced by systemic administration of opiate agonists is at least partially mediated by a direct action upon the midbrain periaqueductal gray. The~e is evidence that this intrinsic analgesia system requires a descending pathway to the spinal cord for a major part of its action. Early studies indicated that the action of morphine at spinal levels depends on descending inf!uences from brain stem |33,58]. Stimulation of PAG sites effective for analgesia inhibits spinal dorsal born neurons responsive tc noxious stimulation [48]. Furthermore, the analgesia due to FAG stimulation [5,8] or systemic opiate administration [5,8,30] is markedly reduced caudal to midthoracic les%ns of ~he spinal dorsolateral funiculus (DLF). Although the analgesia elt c!_$ed by electrical stimulation of FAG or by systemic administration of opiates depends on descending connections from brain stem to spinal cord, direct connections from midbrain PAG to spinal cord are ,,~par~e in the rat and have not been observed ~n the ca~ [7,37]. Thus midbra~n effects upon ~he spinal cord require an intermediate link. The evidence for involvement of serotonin (5-hydroxytryptam~n~, 5-HT) in opiate [ ~6 ~ ! ,~nd stimulation-produced [ 1,29 ] analgesia raises the possibility that th~s H~k from FAG to cord is serotonergic. The fact that most spinal 5-HT arises ~rom neurons in the nucleus raphe magnus of the medulla {I~RM) [/~4] prompted studies on ~he role of ~he NI~M in opiate- and stimulation-produced analgesia. Lesions of NRM b~ock opiate analgesia [54] and electrical stimulation of NRM produces potent analgesia that is reversed by the opiate antagonist naloxone [49]. There is a direct and specific projection from NRM ~hrough the spinal DLF to dorsal horn neurons sensitive to noxious stimulation [ 5,6]. Furthermore, electrical stimulation of NlgM inhibits these neurons by a pathway in the D L F [5,9,21,88]. Thus~ the analgesia produced by electrical stimulation or by opiate injection in P A G is appaioently mediated by NP~M projections to spinal cord. The present study supports this hypothesis by demonstrating a population of neurons in NP~M projecting to spinal cord ~hich is exci~ted by electricalst]mulna;ion of the P A G and by opiate agonists administer,~d sys.temicaHy or by local m~ec~=on " " ~ into midbrain sites.

335 METHODS

Some

procedure~ used in this series of experiments

have been previously

;a~ied out to determine the analgesic L or elect~cal stimulation of the mid° stainless steel guide cannula was ima 30-gauge (0.3 ram) microinjection synnge needle projecting several millimeters beyond the guide c~mula tip to the target site. Similarly, a stimulating electrode~ a wire insulated except at the tip, could be inserted. The guide cannula then served as ground~ Repeated trains of electrical stimuli or microinjection of the opiate agonist etorphine produced a reduction of behavioral responses to noxious stimuli (alligator clip or pinch of paws) and elevation of the threshold of the jawopening reflex to tooth pulp electrical stimulation [48,49]. In these 6 cats, the same chronic injection cannula and midbrain stimulating procedures lent acute experiments. weighing 2.5--3.5 kg were either decerebra~ed temporary halothane anesthesia or were mainrained under chloralose-urethane anesthesia (40 mg chloralose and 200 mg urethane pei ~ kg in saline i.p.). Preparations were paralyzed (intermittent i.v. gallamine triethiodide) and artificially respired. Trache~ carbon dioxide and carotid arterial blood pressure were continuously monitored~ recorded and maintained within physiological range. In animals not already hnpiar~ted~ a stereotaxically placed cannode was used for microinjection and stimulation (Fig. i). Thi~ consisted of tbree 30-gauge needle cannulae~ through which solutions of drugs or saline could be delivered~ and a pair of b~e tip wire electrodes~ cemented together into a 1 mm diameter a~ayo Following occipital craniectomy~ the caudal vermis of the cerebelb.~m was removed by suc~;{on. Electron penetrations were made through the median suicus of the floor of the fourth ventricle. Microelectrodes were filled with pontamine blue and potassium acetate (impedance 4--8 Ma)o Recording sites were marked by eiectrophoretic deposition of dye (Fig. I). Stimulation through pairs of silver ball electrodes placed bilaterally on t~::~ dorsolateral aspect of the lower cervical spinal cord was used to identify sp! nal cord projecting cells {Fig. !). Cells were accepted as projecting to spinal cord only if they responded at fixed latency to electrical stimulation of one DLF and if collision was observed between this fixed la~e.~cy spike and one or~hodromically elicited (Fig. 2A) [23~. D~fficulties in demonstrating collision would exclude two types of projectin~ cells from our s~mple" these i~ which orthodromic ~pikes could not be elicited and tho~e with i!ong ~tency ~ activity. antidromic responses ~ d high rate~ of ~pon,~eouo " A range of intense somatic stimuli was used to te~t the response of the~e cel!~ to noxious inputs. These included pinching a fold of skin with a homostar or biting it with a toothed forceps~ manually squeezing some ....~;~

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Fig. 1. Experimental arrangement. In a preco~licularly dece:.+ebrate and decerebellate preparation, a micropipette recording electrode penetrates thrct~gh the median sulcus. Electrophoretic deposition of dye indicates the rec~rding site of a celt in the nucleus raphe magnus just dorsal to the compact mass of ceils of the nucleu~ raphe pallidus and just rostral to the level of the inferior olive (I0). Pairs ~f stimulating ~lectrodes placed bilaterally on the dorsolateraI surface of the lower cervical spinal cord antidromically activate the cell's descending axon. A eannode inserted i n t o tt~e periaqueductal gray matter ( P ~ ) allow~ testing of the effects on the cell of local d r u g injection~:~nd electrical sf~imulation. Other abbreviations: P, pyramidal tract; SC, superi~r collicMus; IV, fourth ventricle; Vii, facial nucleus.

ture or radiantly b.eating a patch of ~haved, bl a~kened ~ ~ ' to more than ~km 45°C (monitored with a thermistor probe). The latter stimulus was usually tested on the lower bsck. Noxious s~imuli, particularly radimit heat, occasionally produced a rise in the blood pressure. A cell was judged to respond to noxious stimuli only if its response preceded or was not correlated with a rise in blood pressure.

After isolating the action potential of a raphe neuron, a:c~ivity was fed through a window discriminator rand c~mverted by a trigger to puNes° which drove a radiometer whose output, along with ~hat o f the blood pressure moni,~or, was continuo~Js!y recorded° D r u g s ++ere administered either by local mio

337

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Fig, 2. A: ideh:~ification of a raphe.spinat neuron by antldromic activation and co}}ision of Ifi each trace the two positive (up) deflections are artifacts of cervical cord stimulation p A and 800 p s e c via a pair of solder bai! e l e c t r o d e s ) a n d are separated b y 1 m s e e i the t o p ( a n d b o t t o m ) trace, t w o negative action potentials follow, the earlier o n e a t a latency of ! , 8 msec indicatieg a conduction velocity over the 75 m m separating the sites of 42 rn/sec. The later potential is s o m e w h a t m o r e d e l a y e d by the s u b s e q u e n t refractory period, a p h e a o m e n o n we have also observed in spin0retieular n e u r o n s [37 ~ When in the second trace i sec later a spontaneous action potential precedes the paired stimuli, no spike follows the earlier stimule~: it has bee~ obliterated by collision with the spontaneous on~. The la~er spike, not now falling within a r e f r a c t o r y period, follows the later stimulus at 1.8 msee latency. One s e e ~ d later in the int~..rvenes: and the situation is exac~iy as in the !~ir:~t Th ati e a : t mu us at fixed Orthodromicatiy evoked r e c o r d i n g : tO t h e stimulating ~,,iite, in t:his is excited at rather long 1stoney by a train o f 4 s~mitar stimuli:delivered :to ~he PAG.

cromjec~1on intQ ~ d b r a m rotes or through a juguler venous catheter. ~iffee~s on la~eneies from i to 3 rain following d~ag injection. To q u ~ t i g a ~ of a drug injection, pulses were counted and the mean ra~e in the period from 3 go 8 rain after: injection was compezed with preceding injection° This local microinjection~ Such only effects seen follow° mg ~ontro.. mjecSmns of ~so¢om~ saline°

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RESULTS

Data reported here are derived from 6 chronic and i 7 a c u ~ e x p e r i m e n t , Onty data from neurons recorded within the borders of nucleus rapae mag!i 2::(iii17:! nus Opiate e¢'fects ......... : The responses *~o systemic or to local midbrain injection:of opiate agOnis~

and antagonist drugs were studied in up to 4 eelIs cells studied were selected for large, stable and well isoiated action ! potentials and, in experiments in which the cord was stimulated, for spinal projeetiona A c t i v a t i o n o1" N R M n e u r o n s by midbrain injection o f opiates

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In 8 experiments opiates were mieroinjeeted i cats by placing a 30-gauge needle through the cannula. Analgesic effectiveness was assessed by an increase of the jaw-opening reflex (JOR) and by decreased behavioral response to p~ehing the tail and hind paws with toothed forceps, Measurable incre~es 6- ~ R . threshold w e r e observed with 4--10 pg etorphine and were reversed by i n ~ "~ , peritoneal injection of 0.1 mg naloxone hydroehloride. The onset o, anal, gesia was severa! minutes after midbrain injection of etorphine; thus, spread to adjacent neural structures in the midbrain cannot be ruled out, However, since the doses used were much smaller than required to produce anNgesia by systemic administration, spread to disgan~ sites (e,g. cortex, spinal cord or periphery) cannot account for the analgesia. Systematic mapping of midbrain sites sensi;;ive to etorphine was not carried out; however, most injec-

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Fig. 3. Positions of Ni-~@d cel]a located by d:~'e ma,°ks. ,14 dots ( e ) o n and to the right of midiine indicate projecting cells,: 20 crosses { + ) l e f t :~f mid!in:e,

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drug tested. All celts were the most posterior seetion, w h i c h lay jugt dorsal eo :the e a ~ e ~ composing the nucleus raphe palHdus.

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In9 e ments N P ~ M neurons were tested for projection to spinal cord and were by the fixed-latency and collision criteria to prolect to cer~ica! spinM cord. T h e ~ locations are shown m F~g. 3 (ruled mrcles). The conduction velocities of these ceilsrange from 12.5 to 6~. ~ m/see with an average of 30/8 m/sec (Fig. 4). Sampling biases due ~o rather large (5--10 Mf~) electrodes, h~gh a x o n M threshold, or other factors m a y be responsible for the absence of cells with low conduction velocities.

were tested by electrical tM gray. In all 7 experiments raphe-spinal-cells were found which received an excitatory input from

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the midbrain (Fig. 2B). Stimulus parameters used were 1--4 ! 0 0 , 0.2 msec pulses at a f r e q u e n c y of 200 Hz. Of 2 0 raphe,spinal e e l s tested, 15 were excited by midbrain s t i m u l a t i o n and 5 were unaffected. N o n e were inhibited. The e xc i t a t i on ~)roduced by PAG stimuiation was weak and occurred at a variable was r e q u ~ e d to p r o d u e f o u n d between d e e e r e b r a t e u n a n e s t h e t i z e d a n d cMoralose-urethane parations with an intact mesodiencephalic region.

Opiate effects on raphe-spinal neurons Some effect of opiate agonist was observed in 13 o f 17 a d e q u a t e l y tested raphe-spinal n e u r o n s (Table IA). Ten o f the 13 were excited and 3 inhibited by opiate agonist. I n t r a v e n o u s n a l o x o n e reversed the excitation in 5 cells and reversed the inhibition in 2 cells. In 2 cells the naloxone-reversib!e excitation was produc e d by direct midbrain mieroinjection o f opiate agonist. An example of a raphe-spinal n e u r o n response to midbraia injection of m o r p h i n e is s h o w n in Fig. 5A. In this case, 15 #g o f m o r p h i n e sulfate was injected in PAG. The frequency of firing of the cell increased slowly a n d was only detectable after a latency of several minutes. In contrast, the activity was a b r u p t l y t e r m i n a t e d by 0.4 pg n a l o x o n e h y d r o c M o r i d e administered at the same midbrain locus. The firing rate of the cell was n o t correlated with blood pressure changes.

TABLE

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RESPONSES OF OPIATE

OF THE AGONISTS

ONGOING ACTIVITY AND NALOXONE

OF

Agonist drugs, routes, and dosages include morphine

NRM

CELLS

TO MICROINJECTION

sulfate systemically (I--3 mg/kg) or

locally (3--15 pg) and etorphine hydrochloride locally naioxone h y d r o c h l o r i d e was administered either systemically (2 local!~ Local injection was made in a volume of I onggi~g activity of cells are characterized as excitatory spike-counting procedure Aescribed in the text, not cle~ly responding to agonists or naloxone Table [B are classified 49 NRM eel1: upon which at least cells 15 were demonstrate~ to project t o the spinal cord and are shoWn separately in Table IA. IB. All NRM cells

IA. Rapne-spinal cells

Naloxone

Naioxone

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Occasionally a previously silent ceil began to fire at high frequency follow° ing e p i ~ a ~ n i s t injection -:'Fig. 5C). In general, excitatory effects of opiate :: su e d m osed on some spontaneous background discharge and agomstswere pP . _ ~ ~. accelerated to a peak frequency at approximately 10 15 min. Innibitory res ~ oXone were generat~ ~ .~r o n s e t a t the doses used. S u m m a ~ o f opiate actions on all N R M neurons The effect o~ opiates on a total of 62 Nl%M neurons was studied: the 17 " i ussed above; i 6 which could n o t be antidromicaliy raphe- ~ neurons d.sc .... .... activated; ~d~ 29 t h a t were not tested_ . for. spinal.~_~projection.~^~.~In 49 of these

When agonist or antagonist effects were present, t h e y ranged from very marked to barely detectable. Many cells were recorded only long enough to study the effect of a single injection. However, in most cases, responses were i to both a onist and antagonist and in some cases, to both systemic observed ..... g .. ' . " " er and tocal i ~ c t i o n . In Table tB are classified all 49 cells affec~ea by e!th ........

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morphine sulfate (3 ttg, 5 t~g and l b gg), ann ± c ~ by 2-D-Ala met-enkephalin (10 gg). Only one agonist drug was tested by midbrain injection for each NI%M celI. Systemic injection of morphine sulfate was excitatory in 7 neurons (average increment 64%). The average systemic dose of morphine sulfate was 1.4 mg/kg (range 0.94--2.2). This dose has been shown to be effectwe for analgesia in the c~at [46]. When excitatory e ~ c t s to agonist were observed, syst .mic naloxone usuall.v produced a dramatic drop in activity (Fig. 5A)o The average systemic . . . . . . . . . . . .

of

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produced a 13% reduction owed n aloxone,reversible inhibition of :firing when a either locally or systemically (Table tB)o In one neurons were recorded simultaneously and showed " t administrag rate to opiate agonist and antagoms bity was inversely correlated during spontaneous ~seonse to peripheral stirnulatlo °

Ofher factors affecting N R M neurons °~ ?n correlated with blood pressure {BP~ " {a ~elatlon was seen, increased r s wet IB includes 3 neurons whose firirlg was d antagonist. In these c e i i s~ ~~ n e ~,~ew~ w a ; ~ a

342

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j Fig. 5. Effects of opiate injection on the ongoing activity o f 3 different NRM cells. On the upper line of each record, ticks occur at 1 rain intervals and bars represent periods of drug injection. N u m b e r s represent doses in p g given in 1 p| volumes by local midbrain raicroinjection or in p g / k g when administered intravenously. The letter M repre,,~ent~ morphine sulfate; E, e t o r p h i n e hydrochloride; and N, naloxone hydrochloride. On the raiddle line of each record, a rateraeter registers spike frequency, calibrated at the right, in 20 Hz intervals. The carotid m-terial blood pressure is m o n i t o r e d on the lower line and is calibraLed go the right in intervals of 100 rare Hg. The spinally projecting cell in record A was excited at long ]aLeacy by local raorphine :~z~icroinjection and rapidly silenced by naloxone raicroinjec~ion at the same raidbrain site. Some rainutes later ongoing activity resumed. LaLer in this record Lhe arterial cannula becarae clogged and was cleared. In B the cell was apparently depressed by local e t o r p h i n e raicroinjection and the effect was reversed by systemic aa!oxone injecLion, but the correlation of its activity with blood pressure changes is significant° ©piates had no measurable effecL on the average rate of activity of the cell in record C. Shortly after local etorphine raicroinjection, another, prev:~msly silent cell, became active and was subsequently windowed o u t (underlined).

343 steady increase or decrease in rate which was correlated with a progressive physi rA change in the state of the preparation, e.g., the depth of a~esthesia or in the degree of neuromuscular block. drug-induced BP hanges in cell fir........ 3rain injection of administration of agonist. In llof the 14 ceUs showing agonist-induced naloxone-reversible rate increases, the increase could be clearly dissociated from BP changes. This was the case for 4 of the 5 raphe-spinal neurons showing naloxone-reversible excitation b y opiate agonist. It should be stressed that since systemically administered morphine usually produces a drop and naloxone, an elevation, in BP, an indirect action of these drugs by means of BP effects upon cell firing would tend to mask excitatory effects of opiate agonists as well as the naloxone-reversibi!ity of this excitation. In sum, while cells exhibiting both excitation and inhibition to opiate agonist are present in the NRM, the direct effect of agonist injection, not mediated by BP changes, is more often excitatory.

Receptive fields Response properties were adequately studied in 35 of the 68 raphe-spinA ceils. The range of responses and receptive fieid sizes was similar in decerebrate and anesthetized preparations, l~esponses were transient and rapidly adapting when light mechanical stimuli were used. To noxious stimuli, responses were sustained and often accelerating. Despite ezten~ive testing, no receptive field was found in 7 of 35 raphe°spina] neurons. Twenty~one of the rema nmg 28 were driven by a brisk light tap anywhere within a }a~°ge fie]d, u~,d [y includ~g ~e ~rum and fl'equen~ly the entire body. Innocuous tactile s ;imu!ii if slowly applied, were seldom effective. Fourteen, or one-half of

the ~sponsive cells, ~ere e~cited by some form of noxious stimulation in some p-- of their receptive field.In 3 cellsinhibition was the only response foun,| to the noxious stimulation. Six cells exhibited a potent excitation to sounds or to rasping lightly on the solid stereotaxic earbars. DISCUSSION

These experiments demonstrate that there is a population of neurons in the nucleus raphe magnus which projects to the spinal cord and is activated by systemic or midbrain injection of opiate agonists and by midb~ain elec° ~xical stimulation. Since iontophoresis of opiates d~ectly onto cat NRM neurons has either no effect or is inhibitory [25~40] and concentrations of opio ate receptor are low in NI~M [ 3,52], it is likely that in the cat, the excitatory

344 effect of systemic opiates on NI~M neurons is indirect. In the rat, there is some preliminary evidence for a direct opiate action on NRM [41,53] (but s of species including t periaqueductal gray (: there are direct anatc trolateral PAG [24,57] and morphine microinjection or ion~phoresis into ventrolateral PAG of the rat is effective in exciting NRM neurons [ 10]. Electrical stimulation of NRM produces both profound analgesia and inhibition of spinal dorsal horn neurons involved in pain transmission [5,21,28~ 68]. Fu~hermore, lesions of NI%M block morphine's analgesic action [54]. Taken together with the present experiments this indicates that at least ps~ ef morphine's analgesic effect depends on the activation, via the midbrain, .of raphe-spinal projections which inhibit spinal pain-transmission neurons. In this study, we recorded from neurons throughout the nucleus raphc magnus. The mean conduction velocity of this population of raphe-spinal neurons was approximately 30 m/sec with a range of 12.5--60.7. This is consistent with our previous study [2]. It is of interest that no axons were found to conduct at a velocity of less than 12.5 m/sec. Although a consistent sampling bias cannot be ruled out, West and Wolstencroft [67] also failed to find spinal projecting neurons with conduction velocities less than 7 m/sec in NRM. On the other hand, they did find raphe-spina] neurons with conduction velocities ranging from 1.25 to 5 m/sec in the adjacent nucleus raphe paHidus. Anatomical data are not sufficient to resolve this question of fiber diameter. DahlstrSm and Fuxe [ 14] reported track:g small (1--2 pro) diameter 5-HT containing axons from the spinal white matter back to the raphe nuclei. They did not observe autofluorescent myelin sheaths around these small diameter fibers and concluded that they m~e unmyelinated. Although it is ~ossib]e that our technique includes a syste:~atic bias against cells with small diameter axons, it appears that the smaller diameter 5-HT contahing axens originate in nucleus raphe pallidus. ]If raphe magnus is a major source of spinal cord serol;onin, these data indicate that many serotonergic fibers are myelinated. NP~M inhibits flexor reflex afferent actions on both segmental motoneurons and ascending pathways° This inhibition descends in the DLF [18]. The raphe-spinal population described in the present work, which must have mye]inated axom~, could contribute tc this inhibitory control. The inhibition of primate spinothalamic tract neurons by stimulation of NRM is mediated at least pgxtially by axons conducting at velocities greater than I0 m/sec and thus mye]inated [68~. Whether the mye!inated raphe-spinal axons that mediate this inhibition are serotonergic is unknown. A significan~ number of NRM neurons are either unaffected or show non° ~:~pecffic{i°e. non°naloxoneoreversible) responses to opiate agonists injected ~ystemica]ly or into midbrain. This finding is consistent with o'~her single unit studies of NRM in anesthetized rat [ 10,!5] and cat [40], although in a

345

multiple u n i t s of NRM in awake, paralyzed rats only excitation by systemic morphine was reported [47]. Our failure to find uniform opiate effects on raphe-spinal neurons may be a result of the decerebration (which may d e s t r o y the anesthesia used. It is also possible terogeneous population. These studies provide important confirmatory evidence for the proposal [43] that there is an intrinsic analgesia system which is activated in the midbram-PAG and suppresses spmal and tngemmat pam-transm~ssm neurons through the activity of neurons in NRM. This system can be activated by systemieally adminNtered~opiates. Under physlologma, eondtt~ons, the action of this system is presumably mediated by endogenous morphine-like peptides such as the enkephalins. Under what circumstances is this endogenous analgesia system activated? The present experiments provide some information on this question° We have demonstrated that in the cat some NRM neurons are activated by both opiate agonists and noxious stimulation. This has also been reported in the rat [41]. Since the NRM4ndueed inhibition of dorsal horn pain-transmission neurons appears t o require repetitive stimulation [21], noxious stimuli, which produce a sustained discharge in NRM neurons, should be effective in activating this inhibitory system. Anatomical studies show that direct input to the NRM from the spinal cord is sparse [24]. There is, however, a large input from. the nearby nucleus retieularis gigangocellularis which does receive a large input from spinal cord [6,24]. A significant number of spinoretieu!ar neurons respond to noxious stimuli [22,23]. Thus, N1ZM could receive excitation from noxious stimuli via the reticular formation or other, less direct afferent links. If some raphe-spinal neurons receive indirect input from the same dorsal horn pain,transmission neurons that they inhibit, then the elements of a negative:feedback loop controlling pain transmission are present. Disruption of this neg~veifeedbaek loop should either lower pain tolerance or exaggerate pain gehaVior. If endogenous morphine-like compounds are involved at some stage in this negative feedback, it should be possible to disrupt it by the administration of an opiate antagonist (e.g. naloxone). Although some studies have reported no effect of naloxone on pain threshold [17,27,44], there are reports that naloxone shortens hot-plate [34] and tail-flick [i2] latency. Fu~herrnore, acupuncture, which is mildly painful, produces analgesia which is reversed by naloxone in human subjects [ 44]. This evidence is consistent ~ t h the hypothesis that there is an endogenous pain-suppression system mediated by endorphins, which can be activated by noxious stimulation. Further study, preferably in awake and unrestrained animals is necessary to elucidate the physiological role of this system. °

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ACKNOWLEDGEMENTS

The authors would like to thank Dr. Mario Meg!io for pm°ticipatfon during some of these experiments. Dr. Allan Basbaum provided critical help in both

346

experimental design and with Drs. A.J. Harris and MA. Dennis helped in the preparation of this manuscript. This research was s u p p o r ~ d by ~ a n t s from t h e NS 11529, NS 70777 and DA0!949. REFERENCES J Akil, H. and Liebeskind, J.C,, Monoaminergic mechanism of stimulatiemproduced analgesia, Brain Rest, 94 (!975) 279--296, .... 2 Anderson, S.D., Ba~baum, A.I. and Fields, H.L., Response of medullary raphe neurons to peripheral stimulation and to systemic opiates, Brain Res., 123 ( 1 9 7 7 ) 3 6 3 - 368. 3 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Res., 124 (1977) 53--67. 4 Balagura, S. and Ralph, T., The analgesic effect of electrical stimulation of the diencephalon and mesencephalon, Brain Res., 60 (1973) 369--379. 5 Basbaum, A.I., Clanton, C.H. and Fields, H.L., Opiate and stimulus,produced analgesia: functiona] anatomy of a medullospinal pathway, Proc, nat: Acad, SCi: (Wash:), 73 (1976) 4685--4(188. 6 Basbaum, A.I., Clar~ton, C.H. and Fields, H.L., Three bu]bospinal pathways from the ro~stral medulla of the cat. An autoradiographic study of pain modulating systems, J. comp. Neuro]., 178 (1978) 209--224. 7 Basbaum, A.I. and Fields, H.L., Bulbospinal systems of the cat: cell location¢~ and spina~ pathways, Neurosci. Abstr., 3 (1977) 499. 8 Basb~um, A.I., Mar~ey, N.J.E., O'Keefe, J. and Clanton, C.H.~ Reversal of morphine and ,~imulus-produc~ d analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 4 3 ~ 56. 9 Beall, J.E., Martin, ~7~.F., Applebavm, A.E. and W~]lis, W.D., Inhibition of primate spinotha]amic tract hey.tons by stimulation in the region of the nucleus raphe magnus, Brain Res., 114 (1976) 328~333. 10 Behbehani, M.M., E~'f~ct of iontophoresis of morphine and naloxone in the periaqueductai gray on the activity of cells in the nucleus raphe magnus~ Neurosci; Abstr., 3 (1977) 475. .... 11 Bennett, C.T., Hulsebos, B.C. and Bevan, T , E , Signal~detection analysis of changes in nociception in the -~onkey following opiate administration to specific brain areaS, Neurosci. Abstr., 2 (1976) 565. :I 2 Berntsoa, G.G. and Walker, J.M,, ]Effect of opiate receptor blockade on pain sensitiv: ity in ~;he rat, Brain Res. Bull., 2 (1977) 157~159~ 13 Cox, V.C. and Valenstein, E.S., At~;enuation of aversive properties of peripheral shock by hypothalamic stimulation, Science, 1 4 9 ( 1 9 6 5 ) 3 2 3 - - 3 2 5 . 14 DahlstrSm, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous systera, Acta physiol, scand,, 64, Suppl. 247 (1965) 1--30. 15 Deakia, J.F.W., Dickenson, A.H. and Dostrovsky, J,O., Morphine effects on rat raphe magnus neurons, J. Physiol. (Lond.), 267 (1977)46--472. 16 E!de, R., Hhkfel~, T.~ Johaasson, O. and Terenius, L., ][mmunohistochemical studies using antibodies to leu-enkephalin" initial observations on the nervous system of the rat, Neuroscience~ 1 (1976) 349--351. ~7 E1-Sobky, A.~ Dcstrovsky, J;Oi and Walli P , D , Lack of effect of naloxone on pain perception in humans, Nature (Lond.), 263 (t976) 783~784, ~8 Engberg, L, Lund:berg~ A. and RyaH~ R.W., Re¢Sculospinal inhibition of transmission ia reflex pathways, J. Physiol. (Lona.)~ 194 ( 1 9 6 8 ) 2 0 1 ~ 2 3 3 , t9 Ervia, F.R., Brown~ C.E. and Mark, V.H.~ Striatal influence on facial lpaia, Confin. aeurol. (Base]~)~ 27 ( 1 9 6 5 ) 7 5 ~ 9 0 .

347 20 Fields, HiL. and Basbaum, A.I., Brains~em control of spinal pain ~ransmiss~on neurons, Ann. Rev. Phys]oll, 40 (1978) 193--221. ~ W ~ ] ~ H.L.. Basbaum, A,I., Clanton, C.H. and Anderson, S.D., Nucleus raphe magnus ino~ons

~75) 118--134, 24 Gallager, D.W. and Pert, A., Afferents to brainstem nuclei (brainstem raphe, n. reticularis pontJs caudal:is and n. gigantocelIularis) in the rat as demonstrated by microiontoph0reticatly applied horseradish peroxidase, Brain Res., 144 (1978) 257 2'75. 25 G e n t , J 2 . and Woh~tencroft, J.H;, Actions of morphine, enkephalin and endorphin on single neurones in ~he brainstem, including the raphe and the peHaqueductal gray of the cat. In: H.W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides~ NorthHolland, Amsterdam, 1976, pp. 217--224. 26 Gol, A., Relief of pain by electrical stimulation of the septal area, J. neuro]. Sci., 5 (1967) 115--120. 27 Goldstein, A., Pryor, G.T., Otis, L.S. and Larsen, F., On the role of endogenous f naloxone to influence shock escape threshold in the rat, ;04. 2 ., Geisler, Jr., G. and Besson, J.M., Effects induced by stimulation of t h e cenl~ralis inferior nucleus of the raphe on dorsal horn iaterneurons in cat's spinal cord, Brain Res., 126 (1977) 355--361. 29 Hayes,, R.L., Newlon, P.G., Rosecrans, J.A. and Mayer, D.J., Reduction of stimuiation-produced analgesia by lysergic acid diethyiamide, a depressor of serotonergic neural activity, Brain Res., 122 (1977 } 367--372. 30 Hayes, R,L., Price, D.D., Bennett, G.J., Wilcox, G.L. and Mayer, D.J., Differential effects of spinal cord lesions on narcotic and non-narcotic suppression of nociceptive reflexes: further evidence for the physiologic multip|icity of pain modu]ation~ Brain Res.~ in press. 31 Hiller, J.M., Pearson, J. and Simon~ E.J., Distribution of stereospecific binding of the potent narcotic analgesic etorphine in the human brain" predominance in the limbic system, Res. Commun. Chem. Pathol. Pharmacol., 6 (197 3) 1052--1052. 32 ~ u h e s J ~ Smithi T W , Kosteriitz, H.W., Fothergil], L.A., Morgan, B.A. and Morris~ g ' " " ..... ides from the brain with potent opiate H.R., Identification of two related pentapept agonist activity' Nature (Lond.), 258 (1975) 577--579. 3 Irwin S, Houde, R W , Bennett~ D R , Hendershot, L.C. and Seevers, M.H., The 3 : ~'i , ~, . . . . " " " " s onses of spinal animals effects of morphine, methadone and mependme on some re p to nocicepfive stimulation, J. Pharmaco]. exp. Ther., 101 (1951) 132--143. cob. J - , ~ e m b l a y , E C, et Colombel, M., Facilitation de r~actions nocicept~ves par 34 f : naioxone chez la souris et chez le rat, Psychopharmacologia (Berl.), 37 (1974) 2t7--223. 35 Jacquet, Y.F. and Lajtha, A., The periaqueductal gray: site of morphine analgesia and tolerance as shown by 2-way cross tolerance between systemic and intracerebral injections, Brain Res., 103 (1976) 591--513. 36 Kuhar, M.J., Pert, C.B. and Snyder, S.H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447--450. 37 Kuypers, H~G.J.M. and Maisky, V.A., l~etrograde axonal transport of hor~eradish peroxidase from spinal cord to brain stem cell group~ in the cat, Neurosci. Let+~., ! (1975) 9--14. 3g Lewis, V.A. and G ebhart, G.F., Eva~ua~ion of ~he periaqueduc~al central gray (PAG) as a morphine-specific locus of action and examination of morphine-~nduced a~d st~mu|ation produced ana|gesia at CO,he,dent PAG loci, Brain i~es., 124 (1977) 283~

348 39 Lineherry, C.Go and Vierck, C.J., Attenuation o f pain reac~ivityby cauda~e nucleus stimu!ation in monkeys, Brain Res., 98 (1975) 110--134o 40 Loba~z, M A., Proudfit, H.K. and Anderscn, E.G., Effects of noxious stimulation, iontophoretic morphine and norepinephrine on neurons in nucleus raphe magnus,Pharr~acol ........... 4~. Lobatz, M of mc rphi Abstr. 3 (1977) 296. 42 Mayer, D.J. and Liebeskind, J.C., Pain reduction h brain: an anatomical and behavioral analysis, Brain 43 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379--404° 44 Mayer, D.J., Price, D.D. and Rafii, A., Antagonism of acupuncture analgesia in man by the narcotic antagonist naloxone, Brain Res., 121 (1977) 360--373. 45 Mayer, D.J., Wolfe, T.L., Akil, H., Carder, B. and Liebeskind, J.C,, Analgesia from electrical stimulation in the brainstem of the rat, Science, 174 (1971)o1351--1354. 46 McKenzie, J.S. and Beechey, N.R., The effects of morphine and petMdine on somatic evoked responses in the midbrain of the cat, and their relevance to ani~lgesia, Electroenceph, clin. Neurophysiol., 14 (1982) 501--519. 47 Oleson, T.D. and Liebeskind., J.C., Relationship of neural activity in the raphe nuclei of the rat to brain stimulatior, L-produced analgesia, Physiologist, 18 (1975) 338. 48 Oliveras, J.L., Besson, J.M., Guilbaud, G. and Liebeskind, J.C., ~havi0ral ~and electrophysiological evidence of pain inhibition from midbrain stimulation ;in the cat, Exp. Brain Res., 20 (1974) 32--44. 49 Oliveras, J.L., Hosobuchi, Y., Redjemi, F., Guilbaud, G. and Besson, J.M., Opiate antagonist, naloxone, strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior), Brain Res., 120 (1977) 221--229. 50 Pasternak, G.W.~ Goodman, R. and Snyder, S.H., An endogenous morphine-like factor :in mammalian brain, Life Sci., 16 (1975) 1765--1769. 51 Pert, A. and Yaksh, T., Sites of morphine induced analgesia in the primat4~ brain: rela1;ion to pain pathways, Brai~ Res., 80 (1974) 135--140. 52 Pert, C.B., Kuhar, M.J. and Snyder, S.H., Opiate rece:[Aor: autoradiogra~.hic localization in rat brain, Proc. nat. Acad. Sci. (Wash.}, 73 (1976) 3729~3733. 53 Proudfit, H.K., Analgesia following microinjectmn of morphine into the nucleus raphe magnus of acutely decerebrate rats, Neurosci. Abstr., 3 ( I 9 7 7 ) 3 0 0 . 54 Froudfit, H.K. and Ar~derson, E . G , Morphine analgesia: blockade by raphe magnus lesions, Brain Res., 98 (1975) 6 1 2 ~ 1 8 ' 55 Reynolds, D.V., Surgery in the rat during electricalanalgesia induced by focal brain stimulation, Science, 164 (1969) 444--445. 56 Rhodes. D L , A Behavioral Investigation of Fain Responsiveness Following Electrical

Stimulation of the Rostral Brain Stern of the Rat, Ph.D. Dissertation, UCLA, ~ s Angeles, Calif., 1975. 57 Ruda, M., Autoradiographic Study of the Efferent Proiections of the Midbrain Central Gray of the Cat, Ph.D. Dissertation, University of ~'env~y!vania, Philadelphia, Pa., 1975. 58 S.~toh, M. and Takagi, H., Enhancemen~ by morphine of the central descending inhibitory influence on spinal sensory transmission, Europ. J. Pharmacot., 114 (19~]1) 60-65. 59 Schmidek, H.H., Fohanno, D., Ervin, F.R. and Sweet, W.H., Pain threshold alterations by brain stimulation in the monkey, J. Neurosurg., 37 (197I)715---722.

60 Simantov, R., Goodman, ~:L,Aposhian, D. and Snyder, S.H., Phylogenetic distribu-

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63 Takagi, H., Doi, T. and AkalkI, A., ivncromJeu~u~, ~ . . . . . . ~"..... e into the media1 part .~ ~-1~a h , ~ l h ~ r r~f.~eular formation in rabbit and rat: inhibitory effects on ~amina V ...... , Opiates --198.

b y intrabetween ~eg flexor 68 V o g t , M., The effect of lowering the 5-h Yaroxy~ryp~amme ~ , ~ . . . . . . . . . . . ~at spina~ cord on analgesia produced by morphine, a. Physiol. (Lond.), 236 (1974) 483--498. 67 West, D.C. and Wolstencroft, J.H., E1ectrophysiotogical identification of raphe spinal neurones in the cat, Neurosci. Left., 5 (1977) i 4 7 - - 1 5 2 . 68 Willis, W.D., Haber, L.H. and Martin, lg.F., Inhibition of spinothalamic tract ceits and interneurons by brainstem s,~imulation in the monkey, J. Neurophysio!., 4-0 (1977} 968--981. "" 69 Yaksh, T.L., DuChateau, J.C. and R u d y , T.A., Antagonism by methysergme a~.~d cinanserin of the antinociceptive action of morphine administered into the periaqueductal gray, Brain Res., 104

i1976) a v-aTa.