Brainstem and spinal pathways mediating descending inhibition from the medullary lateral reticular nucleus in the rat

Brain Re.search.440 (1988) 1119-122 Elsevier

109

BRE 13242

Brainstem and spinal pathways mediating descending inhibition from the medullary lateral reticular nucleus in the rat A.J. Janss and G.F. Gebhart Department of Pharmacology, Collegeof Medicine, Universityof lowa, Iowa City, IA 52242 (U.S.A.) (Accepted 21 July 1997)

Key words: Lateral reticular nucleus; Locus coeruleus/subcoeruleus; Dorsolateral funiculus; Descending inhibition; Stimulation-produced antinoeiception; Tonic descending inhibition

The lateral reticular nucleus I LRN) in the caudal ventrolateral medulia has been implicated in descending monoaminergic modulation of spinal nociceptive transmission. Experiments were undertaken to examine the organization of pontine and spinal pathways mediating inhibition of the tail-flick (TF) reflex from the LRN in rats lightly anesthetized with pentobarbita!. Microinjections of the local anesthetic lidocaine ipsilaterally or bilaterally into the dorsolateral pons blocked stirr~'ulation-producedinhibition of the TF reflex from the nucleus locus coeruleus/subcoeruleus (LC/SC), but had no effect on descending inhibition produced by microinjection of glutamate into the LRN. Thus, adrenergic modulation of the TF reflex from the LRN is not mediated by activation of spinopetai noradrenergic neurons in the LC/SC. The funicular course of descending inhibition produced by focal electrical stimulation in the LRN was studied in separate groups of rats by reversibly (local anesthetic blocks) or irreversibly (surgical transection) compromising conduction in the dorsolateral funiculi (DLFs) at the level of the ce~'ical spinal cord. Bilateral iidocaine blocks in the DLFs significantly shortened control TF latencies and more than doubled the intensity of electrical stimulation in the LRN necessary to inhibit the TF reflex (153 + 29% increase from control); changes in these parameters produced by unilateral blocks of the DLFs were not statistically significant. Ipsilateral or bilateral transections of the DLFs significantly increased the intensity of electrical stimulation in the LRN to inhibit the TF reflex (110 + 24% and 265 + 46% from control, respectively). Neither lidocaine blocks nor transections of the DLFs completely blocked the descending inhibitory effects of electrical stimulation in the LRN. The DLFs appear to carry fibers mediating LRN stimulation-produced inhibition of the TF reflex as well as tonic descending inhibition of spinal reflexes. The results of the present study indicate that (1) adrenergic modulation of t~c nociceptive TF reflex from the LRN does not depend on a tostral loop through the pontine I C;SC, and (2) descending inhibitory influences from the LRN are contained iJ-t,but not confined to, the dorsal quadrants of the spinal cord.

INTRODUCTION I n v o l v e m e n t of spinal a d r e n o c e p t o r s in the m o d ulation of nociceptive transmission has been affirmed by converging lines o; evidence from n e u r o a n a t o m i c , behavioral, electrophysioiogical and clinical studies. The presence of a - a d r e n o c e p t o r s in the dorsal horn of the spinal cord, where p r i m a r y sensory afferent fibers terminate, has b e e n d e m o n s t r a t e d autoradiographically 5°'55 and by radioligand binding studies 26.

dependent fashion 44"~, and norepinephrinc significantly attenuates responses of single dorsal horn nociceptive neurons to noxious stimuli (pinch and heat) or electrical stimulation at intensities supramaximal for activatiop of C fibers 4A3. T h e analgetic actions of a - a d r e n o c e p t o r agonists ate being exploited clinically; intrathecally administered cionidine and systemically administered adrenergic r e - u p t a k e blockers are currently used to treat pare syndromes 8"~'t2.

modulation of nociception has been confirmed pharmacologically: a - a d r e n o c e p t o r agonists and antago-

The medullary lateral reticular nucleus f L R N ) and pontine nucleus locus coeruleus/subcoeruleus (LC/SC) represent potential sources of descending adrenergic i~lfluences which interact with these phar-

nists delivered to the l u m b a r spinal cord raise and lower nocicepfive thresholds, respec6vely, in a dose-

macologically defined spinal recepto," systems. The L R N in the ventrolateral medulla (dorsal to the infe-

The functional relevance of these receptors to the

Correspondence: G.F. Gebhart, Department of Pharmacology, BSB, U,-d~,ersityof Iowa. Iowa City, IA 522-~2. U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Scie nee Pubiishers B.V. (Biomedical Division)

110 rior olive and ventral to the spinal trigeminal nucleus 3°) lies in close apposition to the A1 norepinephfine-containing cell group 31; the LC in the dorsolateral pons overlaps the A6 norepinephfine-cOntaining cell group (see refs. 5, 25, 57 and 58). The A6 cell group has been shown to be the major source of noradrenergic innervation of the spinal cord in the ratZS'57; there is controversy, however, as to whether noradrenergic neurons from the A1 cell group project to the spinal cord. Studies using histofluorescence and microdissection techniques initially suggested that neurons in the ventrolateral medulla contribute to the catecholaminergic innervation of the spinal cord 1°'14"48. Subsequent studies using immunohistochemical techniques, which label neurons containing specific catecholamine-synthesizing enzymes, indicated that norepinephrine-containing neurons from the A1 cell group send projections rostrally, but do not descend the spinal cord ~5.57'5s. However, a recent report suggests that a significant number of catecholamine-containing neurons from the A1 area do project caudally to terminate in the spinal cord 19. The purpose of the present study was to examine the organization of pathways important to descending inhibition of the TF reflex from the LRN. Since spinopetal efferent neurons from the LC/SC project through the ventrolaterai medulla 25,57, electrical stimulation in the LRN could produce the previously documented spinal a2-adrenocepter-mediated descending inhibition from the LRN 15'24by influencing fibers of passage. Descending inhibitioh from the LC in the rat is also mediated by spinal a2adrenoceptors 27. That glutamate microinjected into the LRN also produces an a2-adrenoceptor-mediated descending inhibition 22 suggests that the A1 and A6 cell groups may be capable of independently modulating nociceptive reflexes 15,22,27or spinal nociceptive transmission 23.zs. However, rostral projections from the LRN to the LC/SC have been demonstrated 32 and it is thus possible that the cz2-adrenoceptor-mediated descending inhibition produced by excitation of ceils in the LRN is mediated through a polysynaptic loop: neurons in the LRN activate noradrenergic neurons in the LC/SC which in turn descend the spinal cord and inhibit nociceptive transmission. Two approaches were taken. The first set of ,experiments examined LRN-LC/SC interactions di-

rectly by reversibly blocking the dorsolateral pons with intra-LC/SC microinjections of local anesthetic and testing whether this block affected inhibition of the TF reflex produced by chemical activation of cell bodies in the LRN. The second approach involved blocking or transecting the dorsolateral funiculi (DLFs) at the level of the cervical spinal cord and examining the extent to which stimulation-produced inhibition of the TF reflex from the LRN was affected. The rationale for these studies arose from evidence that spinopetal fibers from the LC/SC descend the spinal cord in the ventral quadrants of the cervical spinal cord, whereas spinopetal noradrenergic fibers from the lateral tegmental area (A1, A5 and A7 cell groups) reportedly descend in the dorsolateral quadrants s. This segregation of descending systems from the pons and medulla extends to a functional level; inhibition of nociceptive spinal neurons by focal electrical stimulation in the LC/SC has been shown to be mediated by fibers coursing through the ventrolateral funiculus of the cervical spinal cord and is unaffected by lesions of the DLF 29"37. Characterization of the funicular pathway mediating stimulation-produced inhibition from the LRN allowed further comparison of descending inhibition from the LRN and LC/SC. MATERIALS AND METHODS

Surgicalpreparation Experiments were performed in adult male Sprague-Dawley rats (300-400 g; Biolab, St. Paul, MN) initially deeply anesthetized with 50 mg/kg of pentobarbital sodium (Nembutal) i.p. Cannulae were inserted into a femoral artery and vein to permit continuous monitoring of systemic arterial blood pressure and intravenous infusion of pentobarbital (see below) and the rat's head was placed in a stereotaxic apparatus. In 13 experiments, the incisor bar was set 3.3 mm below the horizontal plane and a guide cannula (26 gauge, 0.45 mm o.d.) directed at the LRN 40 was lowered into the ventrolateral medulla. The cervical spinal cord was exposed by laminectomy between C 2 and C3, the dura was removed and the spinal cord was stabilized by vertebral clamps rostral to the laminectomy; this permitted transection (n = 6) of the DLFs or placement of two guide cannulae (26 gauge, 0.45 m m o.d., 1.2 mm separation) at the sur-

111 face of the spinal cord dorsal to the DLFs (n = 9). In 6 experiments, the incisor bar was set 10 mm below the horizontal plane so that two cannulae directed at the LC/SC ( A P - 3 . 1 mm from lambda, 2.2 mm separation in the coronal plane, V - 5 to -6) would not rupture the transverse sinus; a guide cannula was also lowered into the LRN (AP -14.5 mm from bregma, L 1.6, V -7). Local anesthetic ointment was applied to all wound margins and body temperature was maintained with a hot water heating pad.

Spinal nociceptive test Rats were maintained in a relatively lightened anesthetic state (corneal and flexion reflexes present in the absence of other complex behaviors) throughout the experiments with a continuous intravenous infusion of pentobarbital (3-6 mg/kg/h). The TF reflex in rats lightly anesthetized with pentobarbital has been characterized 39'47 and found to be qualitatively similar to that in the conscious rat. In the lightly anesthetized state corneal and flexion reflexes are preserved, but changes in blood pressure and other complex behaviors and reflexes in response to noxious pinch or heating of the skin are absent (see ref. 15). In the present study blood pressure lability in response to noxious stimuli and cort, eal and flexion reflexes were monitored regularly to determine the level of anesthesia. The appropriate level of anesthesia was maintained by adiusting the rate of intravenous infusion. The TF reflex was evoked by radiant heating of the ventral surface of the tail. A minimum of 2 min separated TF trials and the same position on the tail was not heated any two times successively; this protocol minimizes tissue damage 39 and results in stable TF latencies (see Fig. 1). Inhibition of the TF reflex was defined as a TF latency (the interval between exposure of the tail to. and withdrawal of the tail from, the h~at source~ of 7 s (2-3 times the base~,ine TF latency), at which time the tail was moved from the heat source to prevent tissue damage. Each inhibition of the TF reflex by supraspinal stimulation was followed at the next interval by a control TF in the absence of stimulation. These methods have been previously described in detail 15"27"39"47.

Intracerebral electricalstimulation Inhibition of the TF reflex was produced by focal electrical stimulation in the ventrolateral medulla in

all experiments; in 6 experiments inhibition was also produced by stimulation in the dorsolateral pons. Stimulation electrodes were 34 gauge (0.15 mm o.d.) insulated magnet wire (Belden, Richmond, IN) inserted through and extending 2 mm beyond the tip of the guide cannula. Indifferent electrodes (anodes) were inserted subcutaneously. Electrical stimulation (constant cathodal current, 100 Hz, 100/~s pulses, 10-50/~A) was started 10 s prior to and continued during heating of the tail until the TF reflex occurred or 7 s had elapsed. This stimulation paradigm has been established experimentally to result in the lowest thresholds of stimulation for inhibition to the TF reflex 1527. The inhibitory threshold was defined as the lowest intensity of electrical stimulation inhibiting the TF reflex in 3 consecutive trials with controis interspersed.

lntracerebral microinjections of glutamate In 6 cxperiments inhibition of the TF reflex was produced by microinjection of S-glutamate (an excitatory amino acid believed to selectively activate neuronal somata ~7) into the LRN. Stimulating electrodes were replaced with microinjection cannulae (33 gauge, 0.20 mm o.d) which also extended 2 mm beyond the tip of the guide cannulae. Monosodium Sglmamate (50 nmol in water, 0.5 #1, pH 6.8; Sigma) was microinjected over a 1-min period using a 1-#i Hamilton syringe attached to a length of calibrated polyethylene tubing (PE-10) and an in-line air bubble allowed the progress of the microinjection to be monitored; the injector remained in place at least 1 rain after the injection wzs complete. TF latencies were determined 1, 3 and 5 min following glutamate microinjection.

bztracerebral microinjections of lidocaine Rew~.rsible local anesthetic blocks in the dorsolateral pons were made in 6 exeriments to examine the role of the LC/SC in inhibition of the TF reflex produced by glutamate microinjected into the LRN. Lidocaine hydrochloride (0.5/A, 4%; Astra) was injected a~;described above into a site in the dorsolateral ports where focal electrical stimulation at low intensities (~<45/~A) produced inhibition of the TF reflex and also 1 mm ventral to this site. Microinjections of lidocaine were made ipsilateral, then contralateral to the stimulation/microinjection site in the

112 LRN (i.e. a total of 4 0.5-#! lidocaine injections were made bilaterally). The TF latency (mean of 3 TF trials within 10 min following the microinjection of lidocaine), the antinociceptive cfhcacy of S-glutamate (50 nmol, 0.5 #1) microinjected into the LRN, and the inhibitory threshold of stimulation in the LRN were determined following ipsilateral (n = 6), then bilateral (n = 4) local anesthetic block of the dorsolateral pons. The efficacy of the local anesthetic block was verified by loss of stimulation-produced inhibition of the TF reflex from the LC/SC with stimulation at least twice the inhibitory threshold determined before the microinjection of lidocaine; rever-

sal 6f the block was verified in each experiment by return of the efficacy of stimulation in the LC/SC (e.g. see Fig. I).

Microinjections of lidocaine into the D LFs Anesthetic blocks in the cervical spinal cord were made in 9 experiments to examine the funicular trajectory of descending axons mediating the inhibitory effects of stimulation in the LRN. Microinjection cannulae (33 gauge, 0.20 mm o.d.) were inserted through and extended 0.5 mm beyond the tip of guide cannulae resting on the dorsal surface of the cervical

ipsi lido A

glutamate

8.

[] []

[]

[]

25

15

15

15

foe

1 A

A

bi lido

[] LRNS (/~A)

glutamate ! glutamate Imrn

~

ZX

LCS (/z~A)

cl 40

v

A 40

t-

as

.= 0 ..J Lz_

2

ol

";," ,]

12,5

6

s

o s t'o t'5 1'2 (8 2'4 io 3's 42

4'8 5'4 6'0 6'6 72

7'8

I--@--Q--O

@--@'-~@

3'2 4'0 4'8 s'6 6'4 7'2 8'4 9'0 9'6 1021081i4120126132

Time (mln) contralateral

ips i lateral

B

Imm

LRN lmm Fig. 1. Representative experiment illustrating the effects of microinjections of lidocaine into the LC/SC on glutamate- and stimulationproduced inhibition of the TF reflex from the LRN. A: filled circles represent TF latency in the absence of electrical stimulation. The open squares and triangles indicate the TF latency during electrical stimulation in the LRN (LRNS) or LC/SC (LCS), respectively; the intensity of stimulation in pA is indicated. Microinjections of glutamate into the LRN (glutamate; 50 nmol in water, 0.5 pl) and lidocaine (0.5#1, 4%) into the LC/SC ipsilateral (ipsi lido) and contralateral (bi lido) to the stimulation/microinjection site in the LRN are indicated, B: the site of electrical stimulation in the LC/SC (filled triangles) is indicated on a representative coronal section through the pans 4°. Two microinjections of lidocaine (0.5 #l, 4%) were made into each side of the dorsolateral pans, one at the site indicated by the filled triangles and a second 1 mm ventral to that site. The estimated functional block produced by lidocaine is represented by the cross-hatched region. C: the site of focal electrical stimulation and microinjection of glutamate in the LRN (filled circle) is indicated on a representative coronal section through the caudal medulla 4". LRN, lateral reticular nucleus; LC, locus coeruleus; Pyr, pyramid; Sp5, spinal sensory trigeminal nucleus; SC, subcoeruleus.

113 spinal cord. Lidocaine hydrochloride (0.2/tl, 4%) was microinjected as described above into the DLF, first unilaterally (ipsilateral or contralateral with respect to the stimulating electrode in the LRN), then into the opposite side to produce a bilateral local anesthetic block. The TF latency (mean of 3 successive TF trials within 10 min following the microinjection of lidocaine) and the inhibitory threshold of electrical stimulation in the LRN were determined following each microinjection. Focal electrical stimulation was tested periodically to demonstrate return of the efficacy of LRN stimulation as an index of reversal of the anesthetic block. Additional experiments were performed to examine non-specific effects of an injection into the DLFs and the specificity of the site of microinjection of lidocaine into the spinal cord. Normal saline (0.2/~i) was microinjected into the cervical D L F in 3 experiments, first ipsilateral and then contralateral to the stimulating electrode in the LRN; microinjections of saline were made into tile same sites as lidocaine had been microinjected following reversal of the local anesthetic block. The TF latency and threshold intensity of stimulation in the LRN to inhibit the TF reflex were monitored following each saline microinjection. Lidocaine (0.4/A) was also microinjected 1 mm below the dorsal surface of the spinal cord into the dorsal columns in 3 experiments; the TF latency and threshold intensity of stimulation in the LRN necessary to inhibit TF reflex were monitored.

dures (n = 3) with the exception that the spinal cord was probed with the forceps so that the DLFs remained intact.

Histology At the conclusion of experiments, the rats were killed with an overdose of sodium pentobarbital i.v. Sites of stimulation and/or microinjection in the brain and spinal cord were marked by electrolytic lesions. The top of the skull was removed anzl the brain was blocked in situ for the 6 animals in which the LC/SC was stimulated in order to preserve the angle at which the head was positioned in the stereotaxic frame. Brainstem and spinal cord tissue was fixed in 10% formalin, frozen and cut in 40/~m coronal sections and mounted on glass slides. Sections were stained with Cresyl violet or hematoxylin-eosin for histological verification of sites of stimulation and/or microinjection or of the extent of spinal transections. These methods have been described in detail elsewhere 15,23.27.29.47.

Statistical analysis All data are presented as mean + S.E.M. Statistical comparisons were made using Student's t-test for paired or grouped data, or a repeated measures analysis of variance (ANOVA) followed by Tukey's Studentized range test for post hoc comparisons; P <~ 0.05 was considered significant. RESt

II

TS

Transections of the D L Fs The funicular course of descending fibers from the LRN was examined further by performing irreversible transections of the DLFs in 6 rats. Prior to transection of the DLFs, a small pledget of gelfoam soaked in dilute lidocaine was applied briefly (2-5 rain) to the cervical spinal cord. The DLFs were cut with a no. 5 forceps at approximately the C,, spinal segment first ipsilateral, then contralateral to the stimulating electrode in the LRN. The effects of transections of the DLFs on the control TF latency and the threshold intensity of electrical stimulation in the LRN to inhibit the TF reflex were monitored 30 min following the transection; the 30-min delay insured dissipation of any effects of lidocaine from the gelfoam. Sham lesions were made by using identical proce-

Microinjections of lidocaine into the dorsolateral pons Electrical stimulation in thc LRN nr LC/SC at low intensities (15.0 + 1.1 and 35.0 + 2.3 I~A, respectively) as well as glutamate (50 nmol) microinjected into the LRN reliably inhibited the TF reflex. Microinjections of lidocaine into the dorsolateral pons reversibly blocked (duration = 52 + 5 rain) LC/SC stimulation-produced inhibition of the TF reflex, but neither control TF latencies (response latency in the absence of electrical or glutamate stimulation) nor descending inhibition produced by glutamate microinjected into the LRN were affected (Figs. 1 and 2). Microinjection of lidocaine into the LC/SC either unilaterally or bilaterally produced no significant changes in mean systemic arterial blood pressure (control = 102 _+ 5 mm Hg). Glutamate microinjec-

114 A I!-

6-

B

"8" 0

>.

0

4 o

R p-2-

glutamate

~me (mln)

c contralateral

ipsilateral

D

LRNS Threshold

(~A)

pre-Iido

15.0 ± 1.0

ipsl-lido

15.5 ± 1.0

bi-lido

16.0 ± 0.5

recovery

16.0 ± 0.~

LC/SC Threshold

(~A)

35.0

± 2.5

>70 >70 55.0

± 5.0

Fig. 2. Summary of effects of microinjections of lidocaine into the LC/SC on glutamate- and stimulation-produced inhibition of the TF reflex from the LRN. A: time course of the effect of glutamate (50 nmol, 0.5/~l) microinjected into the LRN on TF latency (abscissa) before (filled circles) and following ipsilateral (open circles) and bilateral (open squares) microinjections of lidocaine into the dorsolateral pons. TF latency at time C represents the control TF latencies (the mean of 3 trials performed prior to the microinjection of glutamate, which is indicated by the broken line). Sites of focal electrical stimulation in the LRN (B) and in the LC/SC (C) are indicated on representative coronal sections through the caudal medulla and pons, respectively~. Two microinjections of lidocaine (0.5/~l, 4%) were made into each side of the dorsolateral pons, one at the stimulation site (filled triangles) and a second 1 mm ventral to that site. The estimated functional block produced by lidocaine is represented by the cross-hatched region. In two rats, lidocaine was microinjected only into the LC/SC ipsilateral to the microinjection/stimulation site in the LRN; in the 4 remaining rats, both ipsilateral and bilateral blocks of the LC/SC were studied. See caption to Fig. 1 for abbreviations. Pr5, principal sensory trigeminal nucleus. D: threshold intensities of focal electrical stimulation necessary to inhibit the TF reflex from the LRN (LRNS threshold) and LC/SC (LC/SC threshold) prior to (pre-lido) and following microinjections of lidocaine into the ipsilateral (ipsi-lido) and contralaterai (bi-lido) ports with respect to the stimulating electrode in the LRN; reversal of the effects of lidocaine are also reported (recovery).

tion into the LRN transiently (<10 rain), but significantly decreased mean arterial pressure (31.7 + 4.5 mm Hg; range = 23-50 mm Hg).

Microinjections of lidocaine into the D LFs Microinjections of lidocaine (0.2/d) into the DLF of the cervical spinal cord either ipsilateral (n = 4) or contralateral (n = 4) to the stimulating electrode in the LRN did not significantly alter control TF latencies or stimulation thresholds in the LRN necessary to inhibit the TF reflex (Fig. 3). Microinjections of lidocaine bilaterally into the DLFs, however, significantly decreased control TF latencies by a mean 0.5

+ 0.1 s (range = 0.3-0.9 s) from the pre-lidocaine TF latency (2.4 + 0.1 s) and produced over a 2.5-fold increase in the intensity of electrical stimulation in the LRN necessary to inhibit the TF reflex (153 + 29% of the pre-lidocaine value; Fig. 3). These effects were of limited duration (52 + 4 min) and were not reproduced by microinjections of normal saline into the DLFs (n = 3) or of lidocaine (0.4 ~1) into the dorsal columns (n = 3; see Fig. 3). Bilateral local anesthetic blocks of the cervical DLFs also produced significant decreases in heart rate (53 + 13 beats/min, range = 30-90 beats/min, baseline = 353 + 16 beats/min) and mean systemic

115 A

8-

"•

6-

r"

o, 4 ,.x

.2

,r7 2

pre

lps!

contro

bl

sol

dc

lmm

cont ra B

ips i

60 .~ i I

777 v

I// /// /// /// /// /// /// /// /// /// /// ///

zl. 40-

0

V) z ,_J

20-

/// ///

/// /// pre

Ipsl contro

bl

sal

de

Imm

Fig. 3. Summary of effects of microinjections of lidocaine into the DLFs on LRN stimulation-produced inhibition of the TF reflex. A,B: A, the control TF latency (mean of 3 successive TF trials) and B. the intensity of electrical stimulation in the LRN necessary to inhibit the TF reflex ( LRNS threshold) arc plotted for various treatments: pre, prior to microinjections of lidocaine; ipsi, following microinjection of lidocaine ((I.2 td. 4% ) into the DLF ipsilateral to the stimulating electrode: contra, following microinjection of lidocaine into the DLF contralateral to the stimulating electrode: bi, following bilateral microinjechons of lidocaine into the DLFs: sal, following bilateral microinjections of normal saline into the DLFs: and de. following microinjection of lidocaine into the dorsal columns. * Significantly different (P ~< ILI)5) from the TF latency or threshold stimulation intensity prior to microinjections of lidocaine (pre) by repeated measures ANOVA followed by Tukey's Studenlized range test. C: sites of focal electrica! stimulation in the LRN 07 = 9) and D, sites of microinjections of lidocaine (0.2!d, 4% ) into the DLF are indicated on coronal sections through the caudal medulla a" and cervical spinal cord, respectively. E: sites of microinjections of lidocaine (0.4!d, 4%) into the dorsal columns 4filled circles) and normal saline ((I.2 ul) into the DLFs (open circles) are illustrated on a representative coronal section through the cervical spinal cord.

arterial pressure (18.5 + 5.2 m m Hg, range = 1 0 - 2 7

Transections of the DLFs T h e s e e x p e r i m e n t s c o m p l e m e n t those of the pre-

m m H g , baseline = 99.5 + 2.4 m m H g ) which persisted 49 _+ 5 min. C a r d i o v a s c u l a r p a r a m e t e r s w e r e

ceding section since the e x t e n t o f surgical tran-

n o t significantly altered by iidocaine blocks o f the

sections, unlike anesthetic blocks o f the spinal c o r d ,

D L F ipsilateral o r c o n t r a l a t e r a l to the stimulating

can be histologically d o c u m e n t e d . T h e extent o f tran-

e l e c t r o d e in the L R N , m i c r o i n j e c t i o n s of saline into

sections of the D L F s are s h o w n in Fig. 4; only those

the D L F s or m i c r o i n j e c t i o n s o f lidocaine into the d o r -

transections restricted to the dorsal q u a d r a n t s o f the

sal c o l u m n s .

spinal c o r d w e r e included in the d a t a analysis.

116 contra

ipsi

6

',tam

lmm Fig, 4. Histological reconstruction of transections of the DLFs (A) and sites of focal electrical stimulation in the LRN (B) in rats which received transections of the DLFs (filled circles) or sham lesions (open circles) are indicated on coronal sections through the lumbar spinal cord and caudal ventrolateral medulla4°, respectively. Histological reconstruction of the extent of transections of the DLF is displayed in coronal sections of the cervical spinal cord; the ipsilateral side with respect to the stimulating electrode in the LRN corresponds to the right side of the sections. In one experiment (6), only an ipsilateral transection of the DLF was made.

Bilateral, but not ipsilateral, transectio~ls of the DLFs significantly decreased control TF latencies by a mean 0.5 + 0.1 s (range = 0.3-0.9 s)from TF latencies prior to DLF transection (2.8 + 0.1 s) or following sham manipulations of the DLF (2.6 _+ 0.2 s). The scatter diagram in Fig. 5A clearly illustrates this; the comparison of TF latencies after ipsilaterai DLF transection with those following bilateral DLF transections (filled circles) are all to the left of the dashed line defining equivalence, whereas comparison of TF latencies prior to transection of the DLF with those following ipsilateral DLF trans, ~'tion (open circles) are distributed around the dashed line. Transection of the DLF ipsilateral to the stimulation electrode in the LRN resulted in a doubling of the threshold intensity of stimulation necessary to inhibit the TF reflex (110 _+ 24% of the pre-transection threshold of 22.5 + 2.6~A), an increase that was significantly different from the threshold intensity necessary to inhibit the TF reflex prior to transection of the DLF (t = 4.80, P ~< 0.05, n = 6), and also from that in corresponding sham-treated groups (t = 2.82,

P ~< 0.q5, n = 9). Subsequent transection of the contralateral DLF further increased the stimulation threshold in the LRN for inhibition of the TF reflex (265 _+ 46% from pre-transection value). The intensity of stimulation in the LRN necessary to inhibit the TF reflex following bilateral transections of the DLFs was significantly greater than that prior to transections (t = 7.99, P ~< 0.01, n = 6), following ipsilateral DLF transection (t = 3.87, P ~< 0.05, n = 6), and that of corresponding sham-treated animals (t = 3.41; P ~ 0.05. n = 9). Note that all points in the scatter diagram in Fig. 5B are located to the right of the dashed line defining equivalence. Even though bilateral transections of the DLFs almost tripled the threshold intensity of stimulation in the LRN necessary to inhibit the TF reflex (i.e. to 76 + 6/~A), they did not completely block descending inhibition from the LRN. The cardiovascular effects of cervical transections of the DLFs were similar to those of anesthetic block of the DLF; bilateral, but not ipsilateral transections of the DLFs significantly decreased both heart rate

117 3- l

C

_L

J

A

J

! J

v

X

@o

.]'

I

@

•

L~ _J

6

0

0

~r

/

2-

0

/

.Q

3 h.

C

~

l-

1-

l / /

0-

i 1

0

i 2

i 3

i 4

pre

TT Lafency offer DLI~-X (see)

ipsi

bi

ipsi

bi

B ,,~

D

I00 1 7

J

90-

DLF-X

J

75-

f

r--i Sham

7

.'Q 0 .Q "D

,~ c" ~-

v

60, @

o rr~

@

40-

20-

S ~

8 o

45-

0

.'"

""

60-

(/) Z Q:: _J

0 o

0

30-

15-

Z 0

~ 0

20'

4'0

60'

8 '0

O-

' 100

pro

LRNS Threshold after DLF-X (#A)

Fig. 5. Summary of the effects of tr,asections of the DLFs on LRN stimulation-produced inhibition of the TF reflex. A,B: scatter diagrams illustrate the effects of transections of the DLF (DLF-X) on the TF latency and the intensity of electrical stimulation in the LRN necessary to inhibit the TF reflex (LRNS threshold). Open circles represent TF latency/LRNS threshold before transection of the DLF plotted against TF latency/LRNS threshold after ipsilateral DLF transection. Filled circles represent TF latency/LRNS threshold after ipsilateral DLF transection plotted against TF iatency/LRNS threshold after bilateral transections of the DLF. C: the control TF latency (mean of 3 successive TF trials). D: the LRNS threshold is plotted for various treatments: pre, prior to DLF transection; ipsk following DLF transection ipsilateral to the stimulating electrode: and bi. following bilateral DLF transections, Data from rats which received transections of the DLFs (n = 6) arc represented by the solid bars (DLF-X), data from rats with sham lesions (n = 3) by the open bars (sham). * Significantly different (P <~ 0.05) from the TF latency or threshold stimulation intensity of the corresponding sham-lesion group and from pre-DLF transection group (pre) using repeated measures ANOVA followed by Tukey's Studentized range test. ** Significantly different (P ~<0.d5) from the LRN stimulation tbrc ~holdof the corresponding sham-lesion group, pre-DLF transection group (pre) and ipsilateral DLF transection group (ip~i). Sites of focal electrical stimulation in the LRN and the extent of DLF transections are shown in Fig. 4A,B.

(32 + 7 beats/min, range = 2 0 - 6 1 beats/min, baseline = 317 + 13 beats/min) and mean arterial

reflex originating from the L R N is mediated by spinal

pressure (12.0 + 5.0 mm Hg, range = 3 - 4 1 m m Hg,

ciated with the AI norepinephrine-containing cell

baseline = 86.0 + 6.0 mm Hg).

group 3t. Although reciprocal innervation between

DISCUSSION

the LRN and the spinal cord has been demonstrated 32"35"36"49,there is confusion and controvers~ as

a,-adrenoceptors ~s'22 and the L R N is closely asso-

to whether any descending projections from the L R N

Interactions between pontine and medullary inhibitory systems Descending inhibition of the spinal nociceptive TF

are

catecholaminergic

(see

Introduction).

The

LC/SC in the pons is one source of descending spinal inhibition that is mediated by a_,-adrenoceptors at the

118 level of the spinal cord27; spinopetal axons from norepinephrine-containing neurons in the LC/SC pass through the ventrolateral medulla '5's7"58. However. LRN stimulation-produced inhibition of spinal nociceptive reflexes and nociceptive transmission cannot be solely ascribed to activation of passing coeruleospinal fibers since both an antinociception and descending spinal inhibition can be produced by selective activation of cell bodies in the LRN by glutamate ~5-2t-23. The problem lies in resolving pharmacological and electrophysiological data, which clearly indicate that the LRN/AI contributes to descending monoaminergic inhibition of spinal nociceptive transmission, with the lack of anatomic evidence for monoamine-containing projections from the LRN to the dorsal horn of the spinal cord (see Introduction). This study examined the organization of descending inhibition from the LRN. Retrograde (horseradish peroxidase i) and anterograde ([3H]proline32) neuroanatomic studies, as well as neurochemical and pharmacologic studies 51, have demonstrated projections between the caudal ventrolateral medulla and the LC/SC. The results of the present study indicate that these connections are not involved in descending modulation of a spinal nociceptive reflex produced by activation of cells in the LRN. The local anesthetic action of lidocaine blocks signal transmission in both neuronal fibers and somata; neither ipsilateral nor bilateral iidocaine blocks in the dorsolateral pons affected the ability of glutamate microinjected into the LRN to inhibit the TF reflex. Thus, the dorsolateral pons serves as one noradrenergic source of descending inhibition, but neither cell bodies in the LC/SC nor fibers passing through the LRN are required for descending inhibition originating from the LRN. The use of local anesthetics to create reversible 'le. sions' in the central nervous system has been quantitatively characterized ~6.46.47. The efficacy of the lidocaine block was verified in this study by the time-limited loss of LC/SC stimulation-produced inhibition of the TF reflex. According to functional 46"47and histological38 studies, the radius of diffusion of a 0.5-~1 microinjection is approximately 0.5 mm; using this value, the functional extent of the block was estimated by histological reconstruction of microinjection sites. In all 6 experiments, the block included the LC/SC. The possibility remains that LRN ~ LC/SC

projections are involved in stimulation-produced inhibition from the LRN, but the system must be highly redundant. Either the lidocaine block in the LC/SC did not inactivate a sufficient number of descending inhibitory projections to attenuate glutamate-produced inhibition from the LRN, or neurons in the LRN activate multiple descending inhibitory systems which compensate for the incapacitated pontine contribution to LRN-mediated inhibition. The caudal position of the LRN in the brainstem and the numerous descending projections which course through the ventrolateral medulla made it important to use a selective chemical stimulus such as glutamate. The excitatory amino acid glutamate reliably elevates TF latencies in a volume/dose-dependent and time-limited fashion when microinjected into the LRN 15'22and, in contrast to focal electrical stimulation, will activate only neuronal somata 17. This selectivity was important in these experiments since the role of rostral afferent projections from the LRN in descending spinopetal inhibition from the LRN was focused upon; coeruleospinal fibers implicated in inhibition of spinal nociceptive transmission 1s'27-29'37 project through the ventrolateral medulla 25'57"58 and would be activated by electrical stimulation, but not by glutamate. The conclusion suggested by these experiments that LRN-mediated descending inhibition is independent of a loop through the LC/SC is consistent with reports in the literature. If inhibition from the LRN were mediated by coeruleospinal projections, one would expect inhibition produced from these nuclei to be qualitatively, if not quantitatively, similar. Electrophysiologic23"29'37 and neuroanatomical 5'25'57 studies have, however, shown that inhibition from the LC/SC and the LRN differ in their spinal trajectories (descending principally in the vemrolateral funiculi and DLF at the level of the cervical spinal cord, respectively), and in the qualitative fashion in which they affect the intensity coding of spinal dorsal horn neurons to graded noxious heating of the plantar surface of the hind foot. Focal electrical stimulation in the LC/SC decreases the slope of the stimulus-response function (SRF) while electrical stimulation in the LRN decreases the slope of the SRF and elevates significantly the threshold for neuronal response 23"29. The difference in the qualitative nature of descending inhibition from the LRN and LC/SC suggests that they are not mediated by a final common

119 pathway. Moreover, if the descending inhibition from the LRN required a rostral loop to the LC/SC, one might expect that the thresholds for inhibition of spinal nociceptive reflexes and spinal nociceptive neurons would be equivalent from the two areas, if not greater from the LRN. Instead, thresholds for stimulation-produced inhibition from the LRN are significantly lower for both spinal reflexes and spinal neurons than from the LC/SC (see refs. 15, 23, 27 and 28).

Role of the DLFs in descending inhibition from the LRN Examination of the role of the DLFs in descending inhibition from the LRN served to (1) characterize the spinal organization of descending fibers mediating inhibition of the TF reflex from the LRN, and (2) extend electrophysiological studies in which the role of the DLFs in LRN stimulation-produced inhibition of spinal neurons was established z3. Small volumes of lidocaine (0.2/d) microinjected into the DLFs bilaterally produced a reversible elevation of the threshold intensity of stimulation in the LRN necessary to inhibit the TF reflex. Control experiments indicated that the effect was specific with respect to site (was not mimicked by microinjections of lidocaine into the dorsal columns) and the result of local anesthetic action (microinjections of saline into the DLFs did not have any effect). Fibers from the ventrolateral medulla appear to descend bilaterally in the DLFs at the level of the cervical spinal cord; LRN stimulation-produced descending inhibition was significantly affected only when fibers in both funiculi were compromised by local anesthetic. Similar results were obtained with irreversible transections of the DLF, with the exception that both ipsilateral and bilateral transections of the DLFs produced significant increases in the intensity of electrical stimulation in the LRN necessary to inhibit the TF reflex. Recent electrophysiologicai studies support these reported here; ipsilateral transections of the DLFs at the cervical level of the spinal cord attenuate the efficacy of LRN stimulation-produced inhibition of responses of spinal neurons to noxious heating of the skin 23. The effects of transections of the DLFs on LRN-mediated inhibition of dorsal horn neurons and of the TF reflex are comparable. The apparent discrepancy in the effects of reversible lidocaine blocks

in the DLF and irreversible transections of the DLF is likely due to the volume of local anesthetic used (0.2/A). The small size of the rat spinal cord makes it difficult to contain the extent of local anesthetic block; efforts to limit the spread of the local anesthetic to the dorsal compartments of the spinal cord would also limit efficacy of the block. The shallow position of the DLF may also reduce the efficacy of the lidocaine block as it makes leakage of the injectate onto the dorsum of the spinal cord likely. Transections of the DLF are irreversible manipulations, but complement the use of lidocaine blocks in the DLF as they insure the disruption of the fibers in the funiculi and permit histological verification of the extent of the lesion. Note that increases in the intensity of stimulation in the LRN necessary to inhibit the TF reflex produced by bilateral transections of the DLFs (265 + 46% of the pre-transection threshold of inhibition) were significantly greater than those produced by bilateral lidocaine blocks in the DLFs (153 _+ 29% of the pre-lidocaine threshold of inhibition; t = 2.64, P ~< 0.05), which likely reflect the more extensive lesion produced by cutting the DLFs. It is also noteworthy that neither bilateral lidocaine blocks nor transections of the DLFs fully prevented descending inhibition of the TF reflex by stimulation in the LRN. The intensities of stimulation were increased significantly by both manipulations, and thus the spread of current in the caudal medulla was greater than that associated with stimulation prior to either manipulation. It is likely that the greater amount of tissue thus influenced by electrical stimulation as well as axons from the LRN possibly contained in the ventral spinal white matter, and not examined in the present study, contributed to the failure to completely prevent descending inhibition of the TF reflex by stimulation in the LRN. Depressor effects produced by bilateral iidocaine blocks and transections of the DLFs confirm the efficacy of the transection in this compartment of the spinal cord. Tonic pressor systems descend bilaterally in the spinal cord, converging on the intermediolateral cell column in the thoracic spired cord ~'2°a'-. and both DLF transections and local anesthetic blocks reduced vasomotor tone. Experiments employing transections or local anesthetic blocks of the DLF should be interpreted with caution as these measures non-selectively interrupt

120 numerous descending5'6'29'45 and ascending pathways34.52 implicated in antinociception and nociception, respectively. These interventions may themselves alter the reflexes or responses of the animal to noxious stimuli. In this study transections and lidocaine blocks of the DLF never totally eliminated LRN stimulation-produced inhibition, and to this degree they were selective. The effects of lidocaine blocks and the transections of the DLF were in accord, suggesting that descending fibers from the ventrolateral medulla involved in inhibition of the TF reflex are contained in the ipsilateral and contralateral DLFs. LRN stimulation-produced inhibition of the TF reflex and of responses of spinal neurons to noxious heating of the skin is mediated by descending projections that course in the DLF bilaterally, but which are not confined to the dorsal tracts 23. For example, spinopetal projections from the LRN may also descend in the ventral spinal white matter, as suggested above. Regardless, the accordance between results from studies employing electrophysiological techniques and nociceptive reflex tests suggests that LRN-mediated inhibition of individual nociceptive spinal neurons is relevant to a behavioral end point.

Source of spinal monoamines Given that descending inhibition from the LRN does not depend on a rostral loop through the LC/SC, the origin of monoaminergic fibers mediating centrifugal modulation from the LRN remains in question. Direct spinal projections from the AI catecholaminecontaining cell group may mediate the effect 19. Alternatively, epinephrine-containing cells of the C1 cell group located in the rostral ventrolateral medulla 31 may be activated by stimulation in the LRN to produced inhibition of spinal nociceptive transmission; neurons in the C1 cell group receive afferent fibers from the caudal ventrolateral medulla 4L54, and descend to the thoracic spinal cord 4z'54. Projections from the C1 cell group to the lumbar spinal cord have yet to be demonstrated, however. Spinal serotonin receptors also have been implicated in LRN-mediated descending inhibition 15'22. Although serotonin-containing neu,ons in the caudal medulla reside rostral and medial to the LRN r', projections from the LRN to the nucleus raphe magnus (NRM) and midbrain periaqueductal gray (PAG)

have been demonstrated 33"53. The NRM is the principal source of serotonergic innervation of the spinal cord 5 and raphespinal fibers are thought to mediate, at least in part, descending inhibition from the PAG 3. Inhibition of spinal nociceptive transmission produced by stimulation in the NRM or PAG are attenuated by DLF lesions 3'29'37"45. One might speculate that the serotonergic component of LRN-mediated inhibition is the result of rostral projections to the NRM (or PAG).

Tonic descending modulation Spinal reflexes and neurons in the dorsal horn of the spinal cord are subject to tonic inhibition which is uncovered following transections or reversible cold block of the spinal cord (cf. ref. 59). Bilateral local anesthetic blocks or transection of the DLFs in the present studies significantly shortened the TF latency, which may be interpreted as release of the TF reflex from tonic supraspinal inhibitory influences. Other investigators have observed hyperreflexia or hyperalgesia (shorter latencies to escape o1' respond to noxious stimuli) in rats II and primates 56 following destruction of DLFs. The reported effects of transections of the DLFs on reflex or response latencies are not consistent (see refs. 2 and 43), probably owing to methodological differences: different lesion technique (transections, cold blocks, local anesthetic blocks), different spinal levels (cervical, thoracic), use of different noxious stimuli (mechanical, noxious heat, electrical stimulation), and testing at different times after transecting the DLF. The data from this and other studies indicate that fibers mediating tonic inhibition descend in the DLFs. Electrophysiological studies suggest that descending pathways in the ventrolateral funiculi also carry axons exerting tonic inhibitory influences cn spinal neuronal activity28-37"45. Supraspinal tonic inhibitory systems appear to be diffusely organized, descending in dorsal and ventral compartments of the spinal white matter. Although descending inhibition of spinal nociceptive transmission from the LC/SC is mediated by fibers in the ventrolateral funiculae 3°.38, neither neurons nor fibers of passage in the dorsolateral pons appear to mediate tonic descending inhibition of spinal reflexes since bilateral lidocaine blocks of the LC/SC performed in the present study did not alter TF latencies. In conclusion, descending inhibition from the LRN

121 is m e d i a t e d primarily, a l t h o u g h not exclusively, by

ACKNOWLEDGEMENTS

the D L F s at the level o f the cervical spinal cord. This inhibition d o e s not a p p e a r to d e p e n d on a rostral l o o p t h r o u g h the pontine LC/SC. T h e present study c o m p l e m e n t s similar e l e c t r o p h y s i o l o g i c studies and lends b e h a v i o r a l significance to the d e m o n s t r a t i o n that L R N s t i m u l a t i o n - p r o d u c e d inhibition of spinal dorsal

W e gratefully a c k n o w l e d g e the technical assistance o f Michael B u r c h a m ~nd secretarial assistance of Marilynn Kirkpatrick. This w o r k was s u p p o r t e d by D H H S A w a r d s D A 02879 and NS 19912. A . J . J . was s u p p o r t e d by T32 G M 07069.

h o r n n e u r o n s is m e d i a t e d by fibers c o n t a i n e d in the DLF.

REFERENCES 1 Amaral. D.G. and Sinnamon, H.M., The locus coeruleus: neurobiology of a central noradrenergic nucleus, Prog. Neurobiol., 9 (1977) 147-196. 2 Barton, C., Basbaum, A,I. and Fields, H.L., Dissociation of supraspinal and spinal actions of morphine: a quantitative evamarion, Brain Research. 188 (1980) 487-498. 3 Basbaum, A.I.. Marley. N.J.E., O'Keefe, J. and Clanton. C.H.. Reversal of morphine and stimulus produced analgesia by subtotal spinal lesions, Pain. 3 (1977) 43-56. 4 Belcher, G.. Ryali. R.W. and Schaffer, R., The differential effects of 5-hydroxytryptamine. noradrenaline and raphe stimulation on nociceptive and non-nociceptlve dorsal interneurons in the cat, Brahl Research. 151 (1978) 307-321. 5 Bj6rklund, A. and Skagerberg, G., Descending monoaminergic projections to the spinal cord. In B. SjOlund and A. BjOrklund (Eds.), Brain Stem Control of Spinal Cord MeOzanisms, Elsevier, Amsterdam, 1982, pp. 55-88. 6 Bowker, R M . , Westlund, J.N. and Coulter. J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Research. 226 (1981) 187-199. 7 Ciriello. J., Cavo~on, M.M. and Polosa, C., Function of the ventrolateral medulla in control of the circulation, Brain Res. Rev.. 11 (1986) 359-391. 8 Coombs. D.W., Saunders, R.L., Fratkin, J.D.. Jensen, L.E. and Murphy, C.A., Continuous intrathecal hydromorphone and clonidine for intractable cancer pain, J. Neurosurg., 64 (1986) 890-894. 9 Coombs. D.W., Saunders, R.L., Lachance, D., Savage, S., Ragnarsson, T.S. and Jensen, L.E., lntrathecal morphine tolerance use of intrathecal clonidine, DADLE, and intraventricular morphine, Anesthesiology, 62 (1985) 358-363. 10 DahlstrOm. A. and Fuxc~ K.. Evidence for the resistance of monoamine neurons in the central nervous system. II. Experimentally induced changes i~ intraneuronal amine levels of bulbospinal neuron systems, Acta Physiol. Scand.. 64, Suppl. 247 (1965) 1-36. 11 Davies, J.E., Marsden. C.A. and Roberts. M.H.T., Hyperalgesia and the reduction of monoamines resulting fiom lesions of the dorsolateral funiculus Brain Researo';. 261 (1983) 59-68. 12 Feimann, C., Pain relief by antidepressants: possible modes of action, Pain. 23 (1985) !-8. 13 Flleetwood-Walker, S.M.. Mitchell, R., Hope, P.J., Molonv, V. and Iggo A.. An tt_,-receptor mediates t,hc selective inhibition of nociceptive responses of identified dorsal horn neurons. Brain Research, 334 (1985) 243-254. 14 Fleetwood-Walker, S.M., Coote, J . H and Giibey, M.P.,

Identification of spinally projecting neurons in the A t catecholamine cell group of the ventrolateral medulla, Brain Research, 273 (1983) 25-33. 15 Gebhart, G.F. and Ossipov, M.. Characterization of inhibition of the spinal nociceptive tail flick reflex in the rat from the medullary lateral reticular nucleus, J. Neurosci., 6 (1986) 701-713. 16 Gebhart, G.F., Sandkiihlcr, J., Thalhammer, J.G. and Zimmermann, M., Inhibition of spinal nociceptive information by stimulation in midbrain of the cat is blocked by lidocaine microinjected in nucleus raphe magnus and medullary reticular formation, J. NeurophysioL, 50 (1983) 1446-1459. 17 Goodchild, A.K., Dampney, R.A.C. and Bandler, R., A method for evoking physiologic responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system, J. Neurosci. Meth., 6 (1982) 351-363. 18 Hodge, Jr., C.J., Apkarian, A.V., Steven, R.T., Vogelsang, G.D., Brown, O. and Frank, J.I., Dorsolateral ponstine inhibition of dorsal horn cell responses to cutaneous stimulation: lack of dependen~, on catecholaminergic systems in cat, J. Neurophysiol., 50 (1983) 1220-1235. 19 Hudson, M.E., Fuxe, K., Goldstein, M. and Kalia, M., Spinal projections of noradrc~ergic and adrenergic neurons in the medulla oblongata: new evidence in central cardiovascular control, Soc. Neurosei. Abstr., 12 (1986) 535. 20 Imaizumi, T., Granata, A.R., Benarroeh, E.E., Sved, A.F. and Reis, D.R.. Contributions of arginine vasopressin and the sympathetic nervous system to fulminating hypertension after destruction of neurons of caudal ventrolateral medulla ia the rat, J. Hypertens.. 3 (1985) 401-501. 21 Janss, A.J., Cox, B.F., Brody, M.J. and Gebhart, G.F., Dissociation of antinociceptive from cardiovascular effects of stimulation in the lateral reticular nucleus in the rat, Brain Research, 405 (1987) 140-149. 22 Janss. A.J. and Gebhart, G.F., Spinal monoaminergic receptors mediate the antinociception produced by glutamate in the medullary lateral reticular nucieus, J. Neurosci.. in press. 23 Janss, A.J. and Gcbhart, G.F.. Quantitative characterization and spinal pathway mediating descending inhibition o1 spinal noeiccptive transmisson from the lateral reticular nucleus in the rat. J. Neurophysiol., in press. 24 Janss, A.J., Jones, S.L. and Gebhart, G.F., Effect of spinal norepinephrine depletion on descending inhibition of the tail flick reflex from the locus coeruleus and lateral reticular nucleus in the rat. Brain Research. 400 (1987) 40-52. 25 Jones, B.E. and Yang, T., The efferent projections from the reticular formation and the locus coeruleus studied by

122 anterograde and retrograde axonal transport in the rat, J. Comp. Neurol., 242 (1985) 56-92. 26 Jones, D.J., Kendall, D.E. and Enna, S.J., Adrenergic receptors in rat spinal cord, Neuropharmacology. 21 (1982) 191-195. 27 Jones, S.L. and Gebhart, G.F., Characterization of coeruleospinal inhibition of the nociceptive tail flick reflex in the rat: mediation by spinal a2-adrenoceptors, Brain Research, 364 (1986) 315-320. 28 Jones, S.L. and Gebhart, G.F., Quantitative characterization of coeruleospinal inhibition of nociceptive transmission in the rat, J. Neurophysiol., 56 (1986) 1397-1410. 29 Jones. S.L. and Gebhart. G.F., Spinal pathways mediating tonic, coeruleospinal and raphespinal descending inhibition in the rat. J. Ne,~rophysiol.. 58 (1987) 138-159. 30 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. I. Cytoarchitectonic considerations, J. Comp. Neurol., 233 (1985) 285-307. 31 Kalia. M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic. noradrenergic (A I and An) and adrenergic neurons, nerve fibers, and presumptive terminal processes, J. Comp. Neurol.. 233 (1985) 308-322. 32 Loewy, A.D., Wallach, J.H. and McKellar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 33 Marchand, J.E. and Hagino, N., Afferents to the periaqueductal gray in the rat. A horseradish peroxidase study, Neuroscience, 9 (1983) 95-106. 34 Martin, R.J., Apkarian, A.V., Martini, S.M., Stevens, R.T. and Hodge Jr., C.J., Responses of ventrobasal thalamic units to cutaneous stimuli and the effects of spinal cord blocks of the ventrolateral and dorsolateral quadrants, Soc. Neurosci. Abstr., 12 (1986) 231. 35 Men,trey, D., dePommery, J. and Besson, ,I.M., Electrophysiological characteristics of lumber spinal cord neurons backfired from the lateral reticular nucleus in the rat, J. Neurophysiol., 52 (1984) 595-611. 36 Men,trey, D., Roudier, F. and Besson, J.M., Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase, J. Comp. Neurol., 240 (1983) 439-452. 37 Mokha, S., McMillan, J.A. and lggo, A., Pathways mediating descending control of spinal nociceptive transmission from the nuclei locus coeruleus (LC) and raphe magnus (NRM) in the cat, Exp. Brain Res., 61 (1986)597-606. 38 Myers, R.D., Injection of solutions into cerebral tissue: relatic.:~ between volume and diffusion, Physiol. Behav., 1 (1966) 171-174. 39 Ness, T.J. and Gebhart, G.F., Centrifugal modulation of the rat tail flick reflex evoked by graded noxious heating of the tail, Brain Research, 386 (1986) 41-52. 40 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1982. 41 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Connections between the nucleus of the tractu.: solitarius and the rostral ventrolateral medulla: a poss;ble anatomical substrate of baroreccptor and other visceral reflexes, J. Comp. Neurol.. in press. 42 Ross, C.A., Ruggiero, D.A., ,ioh, T.H., Park, D.H. and Reis, D.J., Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C I adrenaline neurons, J. Comp. Neuroi., 228 (1981) 168-185. 43 Rydenhag, B. and Anderson, S., Effect of dorsolateral funiculus lesions at different spinal levels on morphine analgesia, Brain Research, 212 (1981) 239-242. 44 Sagen, J. and Proudfit, H.K., Effect of intrathecally administered noradrenergic antagonists on nociception in the rat,

Brain Research, 310 (1984) 295-301. 45 Sandkfihler, J., Fu, Q.F. and Zimmermann, M., Spinal pathways mediating tonic- or stimulation-produced deo scending inhibition from the periaqueductal gray or nucleus raphe magnus are separate in the cat, J. Neurophysiol., 58 (1987) 327-341. 46 Sandk0hler, J., Maisch, B. and Zimmermann, M., The use of local anesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block, Exp. Brain Res., in press. 47 Sandkiihler, J. and Gebhart, G.F., Characterization of inhibition of a spinal nociceptive reflex by stimulation medially and laterally in the midbrain and medulla in the pentobarbital-anesthetized rat, Brain Research, 305 (1984) 67-76. 48 Satoh, K., Tohyama, M., Yamamoto, K., Sakumoto, T. and Shimizu, N., Noradrenaline innervat~on of the spinal cord studied by horseradish peroxidase method combined with monoamine oxidase staining, Exp. Brain Res., 30 (1977) 175-186. 49 Satoh, K., The origin of reticulospinal fibers in the rat: a HRP study. J. Hirnforsch., 20 (1979) 313-322. 50 Seybold, V.S., Neurotransmitter receptor sites in the spinal cord. In T.L. Yaksb (Ed.), Spinal Afferent Processing, Plenum. New York. 1986, pp. 117-140. 51 S~monyi, A., Kanyicska, B., Feminger, A., Fekete, M.I.K. and Szentendrei, T., AI/C 1 cell body lesion-induced changes in catecholamine content of brain nuclei, and in drug-induced changes of spontaneous mobility, Biogenic Amines, 1 (1984) 123-131. 52 Swett, J.E., McMahon, S.B. and Wall, P.D., Long ascending projections to the midbrain from cells of lamina I and nucleus of the dorsolateral funieulus of the rat spinal cord, J. Comp. NeuroL, 238 (1985) 401-416. 53 Takagi, H., Yamamoto, K., Shiosaka, S., Senba, E., Takatsuki, K., lnagaki, S., Sakanake,, M. and Tohyama, M., Morphological study of noradrenaline innervation in the caudal raphe nuclei with special reference to fine structure, J. Comp. Neurol., 203 (1981) 15-22. 54 Tucker, D.C., Saper, C.B., Ruggiero, D.A. and Reis, D.J., Organization of central adrenergic pathways. I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord, J. Comp. Neurol.. 259 (1987) 591-603. 55 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a 2 agonist b;:nding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69-101. 56 Vierck, C.J. Jr., Hamilton, D.M. and Thornby, J.I., Pain reactivity of monkeys after lesions to the dorsal and lateral columns of the spinal cord, Exp. Brain Res., 13 (1971) 140-158. 57 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Noradrenergic projections to the spinal cord of the rat, Brain Research, 263 (1983) 15-31. 58 Westlund, D.W. and Coulter, .I.D., Descending projections of the locus coeruleus and subeoeruleus/medial parabrachial nuclei in the monkey: Axonal transport studies and dopamine-fl-hydroxylase immunocytoehemistry, Brain Res. Rev., 2 (1980) 235-264. 59 Willis, W.D., Control of Nociceptive Transmission in the Sp#~al Cord, Progress in Sensory Physiology, ILhl. 3, Springer, Heidelberg, 1982. 60 Yaksh, T.L., Pharmacology of spinal adrenergie systems which modulate spinal nociceptive processing, PharmacoL Biochem. Behav., 22 (1985) 845-858.