Hypoalgesia following microinjection of noradrenergic antagonists in the nucleus raphe magnus

© Eisevier/NOrth.Holiand Biomedical Press

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H Y P O A L G E S ~ FOLLOWING MICROINJECTION O F NORb~DRENERGtC ~ A G O N I S T S IN "HIE NUCL]~US RAPHE MAGNUS:. . . . . . . . . . . . .

D O r A L, H ~ M O N D , RICHARD A, LEVY and HERBERT X, PROUDFIT

Department of Pharmacology, University of [itinO~'~at the Medical Center, Chicago, Ill: 60612 (u.s,A.) (Received 23 January 1980, accepted 14 May 1980)

SUMMARY

The influence of the noradrenergic input to the nucleus raphe magnus (NRM) on the capacity of this nucleus to modvlate nociceptive threshold was investigated in r a t by microinjection of noradrenergic (NA) antagonists in the NRM. Tb_e distribution of NA terminals associated with the NRM was visualized histochemically using a glyoxylic acid-induced fluorescence technique. Microinjection of 5.0 and 10.0 /~g phentolamine at sites within the NRM, which was found to be densely innervated by NA terminals, produced a dose-related hypoalgesia as assessed by both the taft flick and h o t plate tests. Microinjection of these doses at sites close to yet outside the NRM, in exeas less densely innervated with NA terminals, was either ineffective or produced hypo~gesia only after a substantial delay. Similar results were obtained following microinjection of 10.5 #g azapetine, another NA antagonist. - tribution of NA terminals associated ~ctive sites f o r hypoalgesia in this area Lsed by blockade of the NA input to the . those cells of the NRM involved in the hold are themselves subject to a tonic, inhibitory .~A input, the suppression of which produces hypoalgesia.

INTRODUC' :ION

The nuc teus raphe magnus (NRM), located on the rnidline of the rostral '.end in the dorsolateral funiculus rn of t h e spinal cord, particularly e-ddence that the NRM modulates

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the nociceptive threshold. Lesions of the N R M resultin hyperalgesia [41,43] suggesting that this nucleus may tonically inhibit the transmission of nociceptive information. Conversely, eleci,vicalstimulation of the N R M causes hypoalgesia [37,39,43] and also selectivelyinhibitsthe response of dorsal horn neurons to noxious stimuli [5,15,16,20], In addition, the response of spinothalamic tract cells to volleys £n A~ afferents is inhibit~-~lby N R M stimulation more extensively than is the response of these cellsto volleys in larger diameter afferents [49], Lesions of the dorsolateral funiculus markedly attenuate the inhibitionof dorsal horn neurons [5,16] and spino, thalamic tract cells [49] produced by N R M stimulation. This finding suggests that these effects of N R M stimulation ~ e mediated by its projections to the spinal cord. The involvement of these descending fibersis further suggested by the inhibition of dorsal h e m neuron responses to noxious stimuli produced by iontophoresis in the dorsal horn of serotonin [24,26,44], a possible transmitter released by raphe-spinal fibers [10,11,38]. Together, these studies suggest that activation ~f the N R M suppresses the transmission of nociceptive information via itsprojectionsto the dorsal horn. Despite the above evidence that N R M activitymodulates the nocicepfive threshold, littleis known about the neuronal inputs which, in turn, modulate the N R M . Biochemical [45,48] and histochemical fluorescence studies [9, 17] have indicated that the N R M receives a noradrenezgic (NA) input. The depression of N R M cell firingproduced by iontophoresis of norepinephrine in the N R M suggests that this N A input may be inhibitory [31]. Moreover, Lovick etal. [33] have suggested that some N R M cells may be subject to a tonic inhibitory influence,as antidromic invasion of the soma of these raphespinal neurons could be observed only during depolarization of the soma with DL-homocysteic acid. The N R M also projects to the ventral and lateralhorns of the spinal cord [6,11,38] and is known to be involved in the regulation of motor [25,42] and autonomic functions [I]. Thus, an influence of the N A input on N R M cells regulatingthe nociceptive threshold cannot be assumed. In the present study, the involvement o f t M s N A input in NRM;mediated modulation of nociceptive threshold was examined by microinjection of N A ~gonists in the N R M , If the N A input exerts a ~nlc inhibitoryinfluence on the activity of those N R M cellsinvolved inthe production blockade of ~his input by :microinjecfionof ~ A antagonists~ o M d beiexpected to dis' inhibit (activate}the N R M and result in hypoalgesia'A prelim~ary account of some of these resul~;shas been presented [22]. METHODS

Surgery Under pentobarbital anesthesia (Nembutal, 30 mg/kg i,p,)female SpragueDawley-derived rats (220--280 g)were implanted with a 22-gauge stainless steel guide sheath stereotaxic~ly positioned 2~5 mm above the nucteus:raphe magnus: (NRM coO~Linates 3,0, ~1.0, L 0,0;

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g u i d e sheath was secured to the skull with jeweler's screws and dental ac~lic,:: Occlusion of the sheath was prevented by insertion of a 28-gauge styler. Animals were housed individually and allowed 7 days to recover from surgery. Microinjection procedure and drugs Drags were microinjected v i a a 2S-gauge stainless steel injection cannula inserted through and e x ~ n d i n g 2.5 m m beyond the guide sheath..All drugs were delivered Ln a fixed volume (0.5 #1) of 0.9% NaC1 over a period of a b o u t I rain by means o f a gear-driven Hamilton syringe. Fluid delivery was monitored by observing the movement of an air bubble over a calibrated distance in the length of PE-20 tubing which cormcct~d the injection cannula with the syringe. Following microinjection, the caunula remained in place for 75 sec to minimize the flow of drug back up the sheath. The following agents were microinjected into the NRM: the ~-NA antagonists phento!amine hydrochloride (Ciba-Geigy, Summit, N.J,) and azapetine phosphate [7,36] (Hoffman-LaRoche, Nutley, N.J,):, the local anesthetic tetracaine hydrochloride (Sigma, St. Louis, Mo.); and physiological saline (0.9% NaCl). The doses utilized were: phentolamine, 5.0 and 10.0 pg; azapetine, 10.5 #g (equimolar to 10.0 ~g phentolamine); and tetracalne, 4.7 ~g (equimolar to 5.0 ~g phentolamine). The pH of phentoiamine and tetracaine solutions was adjusted to 6.95 +- 0.05 by the addition of 0.1 N NaOH and that of azapetine to 5.95 + 0.05 by the addition of 1.0 N NaOH, Physiological saline was microinjected at pH 6.95 -+ 0.05 and 5.95 -+ 0.05. The first drug microinjected in all animals was the noradrenergic antagonist (phentolamine or azapetine); only one close of one antagonist was tested: in each animal. Control drugs (saline and/or tetracaine) were subsequently microinjected at the same site in random order. An interval of not less than 6 days separated microinjections ot the same site. Only one site was tested in each animal and either 2 or 3 microinjections were made per site. Analgesiometric testing . . . . . The hypoalgesic activity o f each drug was assessed by both the tail flick: (TF) [ 1 , 2 ] a n d r e h o t plate (HP) [51] tests. Animals were tested prior to, a n d at fixed intervals throughout a 2-h period immediately following micro.. injection of drugs. Hypoalgesia was first assessed on the TF test, in whmh a high intensity beam of light was focused on the blackened t~fi. T h e time which elapsed between onset of the light and the reflex removal (flick) of the tail was defined as the tail flick latency (TFL). The average of 3 successive determinations was recorded. The mean TFL prior to drug administra.. tion on 81 occasions in 38 animals was 4.11 ~ec +- 0.11 S.E.M. Following TF determination, t h e animals were immediately placed on a 55°C copper hot p l a t e to w h i c h they were restricted by a plexiglass cylinder. Hot plate latency (HPL) ,was defined as the interval between placement on the plate and a tick : o f ~ e h i n d p a w o r a jump. One determination was recorded. The mean H P L p ~ o r to ~ administration on 74 occasions in 36 animals was

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11.39 sec + 0.71 S.E.M. Animals which failed to respond by 14 sec on the TF or by 40 sec on the HP test were removed, to minimize tissue damage, and assigned the cut-off latency.

Histology After sacrifice by ether overdose and retrieval of the cannula guide sheath, the brain of each animal was removed and placed in sucrose-formalin fixative. 20 #m coronal sections of the medulla were cut. at 100 gm intervals on a c~jostat (Slee) and stained with cresyl violet. The location of the cannula tip (site of injection) in each rat was plotted by projecting the camera lucida image of the section which contained the injection site o n t h e corresponding plate of an atlas of the medulla. This atlas was constructed from camera lucida images of cresyl viole~stalned coronal sections taken at 300 ~m intervals through the rostrai medulla of an unimplanted rat. The distribution of NA terminals in the vicinity of the NRM was also plotted on corresponding plates of this atlas. These NA terminals were visualized histochemically in the fresh frozen brains of 4 unimplanted rats using the glyoxylic acidinduced fluorescence method of De la Torte and Surgeon [13]. 10/~m coronal sections were cut at 100 #m intervals through the medulla and examined with a Zeiss fluorescence microscope (excitation filter BG-12; barrier filters 44 and 50). Alternate sections were stained with cresyl violet for determination of the rostral~caudal plane of section. Thus, the injection site in each animal was located with respect to the NRM and its surrounding NA terminals.

Statistical analysis Saline was used to control for vehicle effects and the effects of repeated testing over the 2 h period. Alterations in TFL and HPL produced by microinjection of phentolamine, azapetine and tetracaine were evaluated with respect to the a~terations produced by saiine when microinjected at the same sites. Two-way analyses of variance were utilized to make Lhese comparisons. Determinations of the onset and duration of action of the drug effect were made by means of the N e ~ a n - K e u l s test for multiple comparisons at the individual time points [27]. The ability of saline itself to alter the TFL and HPL following microinjecfion was determined using oneoway analyses of variance which compared the post-injection latencies to the pre-injection control latency. RESULTS

(1) Distribution of NA terminals The distribution of NA terminals in the vicinity of the NRM was examined in 4 animals and is illustrated for two representative sections of the NRM in Fig. 1. The densest population of NA terminals occurred within the NRM in an area closely restricted to the midline {photograph a; also compare b with c) and extending slightly dorsal to the dorsal margin of the nu-

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P 5.2

P 4.6

Fig. 1. NA terminals in the vicinity of the NRM as visualized by ~he glyoxylic acidinduced fluorescence m e t h o d of De la Torte and Surgeon [13]. Photograph a represents an area 200 pm wide by 180 pm high. Photographs b--e each represent an area 120 gm wide by 180 pxn high. Magnification, × 200. The rectangles on the plates indicate the locations and sizes of the areas photographed. Photograph a represents the square in section P 5.2. The arrangement of the 4 photographs around section P 4.6 {b--e) corresponds to the arrangement of the 4 squares within this section.

cleus. The majority of temdaals in this midline region were organized in clusters. The density of terminals and the incidence of clustering in this area appeared to be greater in the rostral than in the caudal aspect of the NRM (compare c and d with a). Although NA terminals ;~n more caudal sections

9o exten the f clust~ Ros~ d~Ik also: i ciatec nals i less cl The ,

latera observed in the region situated ventral to the NRM and dorsal to the p:~amids.

(2) Location of microinjection sites The site of inject:ion in each rat was assumed to be identical with the location of the cannula tip. An injection site was considered to be "in the NRM" if it overlapped any portion of t h e NRM: Such sites were, as indicated above, located in an area densely populated with NA terminals. S i ~ located in the midline area immediately dorsal t o the NRM, which a l s o contained a dense aggregate of NA terminals, were also considered to be in the NRM. Sites which did not overlap a n y portion of.the NRM, and therefore located in a region only sparsely populated with NA terminals, were considered to be "outside the NRM." Accordingly, microinjection sites in 2 3 of 38 animals were found to be in the NRM; sites in the remaining 15 animals were located outside the NRM (Fig. 2).

(3) Hypoalgesia follo~ing microinjection of NA antagonists The hypoalgesic activity of the NA antagonists phentolarnine and azapebore-mentioned sites, is have occm~ed following elevated by at least two atency calculated for all requirements were that ection and to persist for at least 20 min. The ability of the NA antagonists to produce hypoalgesia was found to be dependent on ~he location of the injection site with respect to the NRM and its NA terminal field. Hypoalgesia was most reliably produced at sites which involved the NRM proper and the area immediately dorsal to the NRM -a r e ~ which c~ntain a high density of NA terminals (Fig. 1 ) . Hypoalgesia resulted from microinjection of NA antagonists at 15 of 22 sites located in the NRM as assessed by the TF ~est and at 18 o f these same sites as determined b y - t h e HP test. In contrast, no hypoalgesia was observed o n t h e TF test following injection at any of the 15 sites located outside the NRM; hypo~gesia was observed at only ~ of I 4 of these sites as assessed on the HP test.

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Fig. 2. Microinjec-~ion sites within and near the NRM at which phentolamine and azapefine were tested for hypoalgesic activity. The distribution of NA terminals in the vicinity o f the NRM is illustrated b y the shading on the topmost representation of each section. The shading designates the location of the terminals, but does not reflect the relative concentration of terminals (see text). The hypoalgesic activity of the NA antagonists at these sites is indicated (without regard to the individual agents) on under]apQing plates at each level. The criteria f o r hypoalgesia are indicated in the text (Results). @, hypoalgesia on both the t~il flick (TF) and h o t plate (HP) tests; ®, hypoalgesia on the HF only; ~,, hypoalgesia on the TF only; o, no hypoalgesia on cither tes~.

The magnitude and time course of the hypoalgesia produced by microinjection of the individual NA aut~onisf~ are described below. As noted in Methods, the significance of the elevation of TFL and HPL detailed h~ the following sections has been determined by comparison of the drug effect to

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TIME AFTER MICF-~OINJECTION (rain) Fig. 3. Magnitude and time course of the alteration in tail flick latency (TFL) produced by microinjection of 5.0/~g :~hento!amine (e), 10.0/~g phentolamine ( i ) , and saline (A). Abscissa: time after microir.jection of drug in rain. Ordinate: T F L in sec. Each point represents the mean + S.E.I~L P, response latency determined 5--7 rain prior to microinjection o f the drug (arrow). A: alterations in T F L f o l i o w i n g microinjection at sites located in the NRM: 5.0 pg phentolamine, n = 7; 10.0 /~g phentolamine, n = 7; saline, n = 13. B: alterations in T F L following microinjection at sites located outside the NRM: 5.C /~ phento|amine, n = 4; 10.0 pg phentolamine, n ffi 3; saline~ n = 7 . * Significantly different from mean T F L following microinjection of saline at the same sites (NewmanKeuls, P < 0.05).

the effect produced by saline at the same sites using a two-way ANOVA. Microinjection of saline at the same sites as the NA antagonists did not alter the nociceptive threshold, as assessed by both the TF or HP tests. This was true o~ sites• located within the NRM (ANOVA, P > 0.05) and also of sites

0.05 a~ all t~ae points). HY~a]gesia Produced ~:bY/micr0injection ° f both: d0~s of phentolamine at sites located m ~e:NRM::was ~so demonstrableon~:theHP ~est~(F!g~i4A) (two ANOV , P < 0!05). A i:si ic, t elevationoc ! mm of injection :of 5~0 pg phentolamine andwas evident~at most of~the stapled

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Fig. 4. Magnitude and time course of the alteration in hot plate latency (HPL) produced by microinjection of 5 . 0 / ~ phent~)lamine (o), 10.0/~g phentolamine (m), and saline (z~). Abscissa: time after microinjection o f drug in rain. Ordinate: HPL in see. Each point represents t h e mean-+ S:E:M. P; response latency determined 5--7 rain prior t o microinjection o f t h e drug (arrow). A : alterations in H PL following microinjection at sites located in t h e NRM: 5.0 p g phentolamine, n = 6; 10.0 p g phentolamine, n ffi 7; saline, n = 12. B: alterations in HPL following microinjection at sites iocated outside the NRM: 5.0 gg phentolamine, n ffi 3; 10.0 #g phentolamine, n = 3; saline, n = 6. * Significantly diff er en t from mean HPL following microiniection o f saline at t h e same sites (NewmanKeuls, P < 0.05).

time points for at least 115 min (Newman-Keuls, P < 0.05 at 5, 10, 30, 50, 60, 90 and 120 min post-injection). The elevation of H P L following microinjection of 10.0 #g phentolamine was also rapid in onset, occurring within 5 min, and was evident for at least 115 rain (Newrnan-Keuls, P < 0.05 at all subsequent time points). Although comparison of mean HPLs following either dose of phentolamine with those fol!owing saline indicated no reversal phento~ine-induced hypoalg~sia at 120 min, the HPL of 3 of the 5 annals which were hypoalgesic follo~ng the 5,0 ~ug dose had returned to the ure~ru~ level at this t~ei However~ none of the 7 animals receiving 10.0

~hen assessed on the HP the p h e n ~ l ~ m e was not limited to sites in the NRM (Figi 4B), Both 5;0 and 10'0 ~ug phentolamine produced a significant eleva tion of f o l l o ~ g microinjection at siteslocated outside the NRM (two way 0~05}i HPLI however, was slow in onset, ) and 10,0 lag ~sequent time ,~pid onset of roinjection of ~tion at sites

m ~ t u d e o r onset latency. Tt 20 m m earlier than did 5.0 a l t h o u ~ the magnitude of the hypoalgesia Was ~he same for b o t h doses in thi~

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(b) Microinject~on of azapetine, Azapethle, also produced hypoalgesia following microinjection in the NRMi Fig! 5 ~ u s . tra~es the effect on TFL following microinjection of 10i5/~g azapetine, a dose equimo~ar to 10.0 ~g phentolamine. A significant elevation of TFL (two-way ANOVA, P < 0.05) occurred within 40 rain of injection ~ d con80 rain ( Newman 0 a n d 1 2: 0 min tmued for at ~east : . , Keuls;P < 0 ,05 at 40, 9 ....... post injection) (Fig, 5A): ~though Comparison o f m e ~ TFLs follo~ng azapet~e with those following saline indicated no reversal of the azapetine' induced hypo~gesia a t 120 rain, 3 of the 6 a n i m ~ w h i c h becamehypoalgesic h a d recovered the~ pre-azapetine TFL at this time~ Microinjection of 10~5 ~g azapetine outside the NRM (Fig, 5B) resulted in a transient d~rease in TFL (t~oway ANOVA, P < 0:05). apparent o ~ y at the 20rain : p o s t : injectionintercal(Newman-Keuls. P < 0..05).

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Fig. 6. Magnitude a n d time course of the alteration in hot plate latency (HPL) produced by microinjection of 10.5 pg azapetine (e) and saline (A). Abscissa: time after microinjection o f drug in rain. Ordinate: HPL in sec. Each point represents the mean -+ S.E.M. P, response latency determined 5--7 rain prior to microinjection o f the drug (arrow). A: alterations in HPL foiiowing microinjection at sites located in the NRM: azapetine, n ffi 8; saline, n = 5. B: alterations in HFL follo~siz~g rnicroinjecfion at sites located outside the NRM: azapetine, n = 7; saline, n = 6. * Significantly different from mean HPL following microinjection of saline at the same sites (Newman-Keuls, P < 0.05).

Microinjection of azapetine at cites in the NRM also resulted in a significant elevation of HPL (two-way ANOVA, P <: 0.05) (Fig. 6A). This elevation was apparent within 20 rain of injection and conti~ued for at least 100 min (Newman-Keuls, P < 0.05 at 20, 30, 40, 60, 90 and 120 rnin post injection). Two of the 6 animals .which became hypoalgesic were observed to have recovered their pre O.05) (Fig. 6B).

(4) Microinjection of tetracaine Tetracaine was raicrc~njected at sites which had been previously tested with phentolamine tc determine whether the hypoalgesia produced by the N A antagonists was the result of a local anesthetic effect.In contrast to the elevated T F L observed after phentolamine and azapetine,4.7 ~g tetracaine,a dose equimolar to 5.0 #g phentolamine, produced a significantdecrease in T F L when microinjected at sites in the N R M (two-way A N O V A ; NewraanKeuls,P < 0,05 at5~I0,20, and ~.0 min post-injection).At siteslocated outside the N R M , 4.7/~g tetracaine produced only a transient decrease in T F L (two-way A N O V A ; Newraan.Kez~, P<~ 0.05 at 5 and 10 rain post-injection). Tetracaine was without eff,~t when assessed by the H P tes~;H P L was ~t~d either ,.~dthinor outside the P :> 0.05 at all time points post-

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(5) Behavioral effects N o gross m o t o r d e f i c i t s w e r e o b s e r v e d f o l l o w i n g m i c r o i n j e c t i o n o f t h e v a r i o u s d r u g s in this s t u d y , althougP, s o m e m i n o r mot;o~ e f f e c t s w e r e a p p a r ent.. F c NRM,

placement of the N A antagonists at sites outside the N R M , a slight ataxia sometimes occurred which became apparent when the animal reared on its hind legs. None of these a~imals appeared sedated nor did they retire to a comer of the cage following microinjection, but rather remained active both in the cage and on the floor. Microinjection of saline at sites within or outside the N R M did not produce any apparent behavioral effects. Tetracaine was occasionally observed to induce circlingin animals that had not previously exhibited this behavior with other agents. In addition, tetracaine occasionally induced an altered posture in which the animal's back was arched and it sat with a hunched posture. These effectswere not correlated with the site of injection. Activity in the cage or on the floor appeared unaltered following tetracaine, DISCUSSION

The major finding of this study is that microinjection of the N A antagonists phentolamine and azapetine at sites within the N R M and its associated N A terminal field caused a reversivle incre~me in nociceptive threshold as assessed by both the tail flick and hot plate tests. Several observations suggest that this hypoalgesia is specific to the action of these agents as N A antagonists. Thus, hypoalgesia could not be produced by microinjection, at the same sites,of either local anesthetic or salinevehicle. This indicates that the hypoalgesia did not reflect a non,specific effect and did not result from repeated exposure to the test stimulus over the 2 h test interval.Moreover, hypoalgesia was produced by two N A antagonists having different physical and chemical properties. The dose-dependent nature of phentolamine's effect, observed with- respect to the magnitude and time of onsetinthe hot plate and ~ail flick test respectively: suggests a receptor-mediated action. There is a possibility that the observed effect of tlaeN A antagonists on uociceptive threshold reflects not a sensory deficitbut a decreased abilityof the an:~nals to perform the end-point response of the analgesiometric tests. However, it is unlikely that the minimal motor deficits observed would account for the profound prolongation in response latency induced by these agents. Furthermore, animals often exhibited substantial hypoalgesla in the absence o ~ ...... --- ..............................

The N3 nists acre, effective hypoalge.¢

97 trast, ~the antagonists generally failed to produce hypoalgesia followir~g injection at sites located close to y e t out~ide tnis radius (Figs. 3, 4, 5 and 6B). In the few instances in which hypoalgesia was produced at outside sites, it was evident only on the HP (Fig. 2). Hypoalgesia was observed on the HP test f o l l a ~ n g placement: o f phentolamine (but not of azapetine) at outside sites. Howe~er~ t h e substantial delay i n onset o f phentolamine's effect contrasts with t h e immediate onset observed on this test following application at sites in t h e NRM. The delay in onset suggests that this hypoalgesia results from diffusion o f phentolamine to the NRM, rather than an action at a site lateral to the midline. The rapid onset of hypoalgesia following injection of the NA antagonists within t h e NRM also suggests that the NRM is the site of action. However, the onset of hypoalgesia produced by azapetine applied within the NRM w a s somewhat slower than that of phentolamine on both analgesiometric tests. The difference in onset time may reflect differences in the physico~hemical properties of azapetine and phentolamine. As indicated above, ~he hypoalgesia evoked by the NA antagonists was most reliably produced fol:t.c:wingmicroinjection at sites in the NRM, where the density of NA terminals,, is very high. In contrast, placement of the NA antagonists at sites outside, yet close to the NRM, where NA terminal density is low, generally did not evoke hypoalgesia (Figs. 1 and 2). This correspondence between the dh~tribution of NA terminals associated with the NRM and the distribution of active sites for hypoalgesia in this area further suggests that the hypoalgesia was caused by blockade of the NA input to the NRM. The present results indicate a functional role for the NA input to the NRM and suggest that this input serves to modulate the activity of those NRM cells involved in the regulation of nociceptive threshold. Blockade of this input by microinjecticn of NA antagonists caused an increase in noci° ceptive threshold, which is associated with NRM activation [37,39,43]. This suggests that the NA input tonically inhibits NRM cells involved in nociception. This suggestion is further supported by the report that iontophoretic norepinephrine will depress firing of NRM cells [31]. Thus, the present results indicate that those cells of the NRM which are ~nvolved in the modulation of nociceptive threshold are subject to a tonic, inhibitory NA influence, the removal of which cruises hypoalgesia. It is not clear, however, if this NA input inhibitsraphe-spina:[ cells directly or does so by activation of an intercalated inhibitory interneuron. An indirect action of a NA input to the nucleus raphe dorsalis, for example, has been postulated by Aghajanian and associates [4,18]. The present study also suggests a possible mechanism for the analgesic action of opiates. ~ e local applica'~ion of morphine to the NRM by microinjection produced hypoalgesia [14,30,40, but see 47] and although iontophoretic::morphine~has n o t y e t b e e n shown to excite NRM neurons [2,21, 50], systemic administration of rnorphine does increase NRM celt firing [3,37], :Furthermore, m o ~ h i n e inhibits norepinephrine release from sthnulated cortical slices [35] and from axon ~erminals in the spinal trigeminal

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nucleus which ori~nate in t h e nucleus locus coeruleus (LC) [46], Thus, ~piates may produce hypoalgesia :in part:by :inhibiting transmitter release from NA nerve terminals innervating the NRM, thereby removing the tonic NA inhibition of NRM cells demonstrated in the present study. The origin of the NA input to the NRM is unknown. Noradrenergic nuclei wh'~ch may contribute such terminals include the LC in the pons and the more: caudauy located nuclei in t h e rneflulla: However, folIowing elec~olytic lesions of t h e LC~:neither NA term~al :density associated with the NRM [29,32], nor the norepineph. fine content of the NRM [29] were altered, F ~ h e r m o r e , no significant pro. jection from the LC was reported: following discrete application of horseradish peroxidase i n the NRM [19]. Lesions :of the LC induced by monosodium.L-glutamate, however, did cause a small decrease in the norepinephrine content of the medullary raphe complex [23]. Taken together, these studies suggest that the LC may contribute at most only a minor component of the NA input to the NRM. The suggestion by Levitt and Moore [29] that the NA input may originate in nuclei A1--3 remains to be investigated. ACKNOWLEDGEMFNTS

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50 Wolstencroft, J.H., West, D.C. and Gent, J.P., Actions of morphine and opioid peptides on neurons in t h e reticular formation raphe nuclei and periaqueductal gray. Ln: R.W. Ryall and J.S. Kelly (Eds.), Iontoph0resis and Transmitter Mechanismv in the Mammalian Central Nervous System, Elsevier/North-Holland Biomedical Press, Amsterdam, 1978, pp. 341--343. he analgesic action of pethidine (1944) 300--307.