European Journal of Pharmacology. 192 (1991) 293-300 :"~ 1991 I-Isevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 001429999100107J

293

[ziP 51643

a~-Adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord J o a n J. K e n d i g , M a a r i t K . T . S a v o l a ~, Scott J. W o o d l e y a n d M e r v y n M a z e Department of Anesthesia, Stanford Unioersitv School of Medicine, Stanford, ('A 94305-5123. U.S.A. Received 28 May 1990, revised MS received 25 September 1990, accepted 2 October 1990

a2-Adrenoceptors mediate analgesia in vivo. The present study explored the actions of the a2-adrenoceptor agonists dexmedetomidine and clonidine on a nociceptive response in isolated neonatal rat spinal cord. Stimulation of a dorsal root generates a slow ventral root potential (slow VRP) at the corresponding ipsilateral ventral root. The slow VRP meets several criteria for a nociceptive response. Dexmedetomidine (10 nM) and clonidine (200 nM) depressed the slow VRP by approximately 80%. Dexmedetomidine's action was approximately linear over the concentration range 0.5-500 nM, whereas clonidine (20 nM-5 ~tM) exerted biphasic effects. The profile of agonist and antagonist effectiveness characterized the receptor(s) as a2-adrenoceptors; the subtype could not be identified as either a2A or a2~. Naloxone pretreatment partially blocked dexmedetomidine's effect, suggesting a possible endogenous opiate involvement. Dexmedetomidine (0.5-2.0 nM) also depressed the VRP evoked by application of substance P to the cord, implicating postsynaptic as well as possible presynaptic actions. At high concentrations, dexmedetomidine (50-500 nM) depressed the monosynaptic reflex, probably through non-a2-receptor(s). Results from the neonatal spinal cord correlate well with those from in vivo analgesia studies. They suggest an important direct spinal contribution to a2-adrenoceptormediated analgesia. a-Adrenoceptor agonists; Analgesia; Analgesics; Clonidine; a-Adrenoceptors; Dexmedetomidine: Spinal cord (neonatal rat)

I. Introduction

a2-Adrenergic agents are receiving increasing attention as anesthetic adjuvants (Kaukinen and Pykko, 1979; Bloor and Flacke, 1982) and analgesic agents (Eisenach et al., 1987; Yaksh and Reddy, 1981; Reddy et al., 1980; Chance, 1986). The prototype a2-adrenoceptor agonist clonidine is in clinical use for both purposes (Ghignone et al., 1987; Flacke et al., 1987; Goldstein, 1983; Gordh and Tamsen, 1983; Tamsen and Gordh, 1984; Coombs et al., 1986; Engelman et al., 1989). Dexmedetomidine, the d isomer of the imidazole medetomidine, is an a-adrenoceptor agonist more potent than cionidine and more selective for a 2- versus a~adrenoceptors (Virtanen, 1989). Dexmedetomidine has been shown to markedly reduce anesthetic requirement in animals (Virtanen, 1989; Segal et al., 1988) and can itself cause loss of righting reflex and response to tail pinch (Virtanen, 1989; Doze et al., 1988). In addition, systemically applied dexmedetomidine is an effective analgesic in animal tests of nociception (Virtanen, 1989).

i Current address: Department of Pharmacology, Institute of Biomedicine, University of Turku, Turku, Finland. Correspondence to: J.J. Kendig, Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305-5123, U.S.A.

The present study was designed to investigate the effects of dexmedetomidine (m a nociceptive response in isolated neonatal rat spinal cord. In response to stimulation of a dorsal root, the isolated superfused spinal cord of newborn rats generates stable electrical potentials that can be recorded at the exit of the corresponding ipsilateral ventral root. Among these are the monosynaptic reflex (Otsuka and Konishi, 1974) and a slow depolarization of very long time course (10-30 s) (Akagi et al., 1985; Garcia-Arraras et al., 1986). This slow ventral root potential (slow VRP) is related to nociceptive neurotransmission. Its threshold corresponds to that of small diameter slowly conducting afferents (Akagi et al., 1985). Because slowly conducting afferent fibers respond to high threshold input and evoke the slow VRP, the slow VRP is referred to as a nociceptive reflex. A slow VRP can be produced by stimulation of peripheral nociceptors (Yanagisawa et al., 1985) and by application of substance P to the cord (Yanigisawa et al., 1982; Otsuka and Yanagisawa, 1988). It is blocked by opioid analgesics (Yanagisawa et al., 1985) and by the substance P antagonist spantide (Yanagisawa et al., 1982; Otsuka and Yanagisawa, 1988). The present study examined and characterized the effects of dexmedetomidine on the slow VRP and on the monosynaptic reflex, and compared dexmedetomidine to clonidine.

294 2. Materials and methods

3. Results

N e w b o r n (0-5 day old) Sprague-Dawley rat pups were anesthetized with halothane or diethyl ether and decapitated. The spinal cord from the midthoracic to the sacral level was rapidly removed, placed in a chamber and superfused with artificial cerebrospinal fluid ( A C S F ) at a rate of approximately 2,5 m l / m i n . The A C S F consisted of (in raM) NaCI 123, KCI 5, N a H 2 P O 4 . H z O 1.2, CaCI 2 2, M g S O 4 . 7 H 2 0 1.3, N a H C O ~ 26, glucose 30, equilibrated with 95% 02-5% C O z to bring the p H to 7.4, and warmed to a temperature of 2 7 - 2 8 ° C as measured by a thermistor in the c h a m b e r near the cord. A suction stimulating electrode was placed on a large dorsal root, most c o m m o n l y the fourth lumbar root, and a suction recording electrode on the corresponding ipsilateral ventral root at its exit from the cord. Stimuli were single square wave pulses 0.2 ms in duration. Stimulus intensity was adjusted to be well supramaximal for eliciting the slow VRP, and frequency was maintained at 0.02 Hz throughout each experiment. In some experiments substance P (2-20 /aM) was directly applied to the cord by pressure ejection from a micropipette positioned near the insertion of the dorsal root. Responses were amplified by a high gain amplifier with a band width set at DC to 30 kHz and monitored on an oscilloscope screen. For measurement and later analysis, responses were digitized, averaged (n = 5), and stored on diskette. Responses to application of substance P were recorded similarly but not averaged. The monosynaptic reflex was measured and plotted for illustrations without further manipulation; slow VRP's, recorded at higher gain, were digitally filtered by a single pass through an RC filter with a 20 ms time constant in order to minimize the faster noise c o m p o n e n t s of the record. Data acquisition and analysis were handled by commercially available software (pClamp, Axon Instruments). M o n o s y n a p t i c reflex amplitude was measured from baseline to peak. Inspection of a sample of slow V R P ' s from 11 preparations showed a modal peak or m a x i m u m at 3 s after the stimulus, and slow V R P amplitudes were measured at this point. Preparations were allowed to equilibrate for 30 min and were accepted if two control measurements 15 min apart showed less than 10% change in m o n o s y n a p t i c and slow ventral root reflexes. U n d e r control conditions responses from such preparations remained stable for 3 h or more. Drugs were made up as stock solutions in distilled water, diluted to the desired concentration in A C S F , and applied to the cord in the perfusate. I)exmedetomidine and atipamezole were the gift of Farmos G r o u p Ltd, Turku, Finland; all other pharmacologic agents and chemicals were purchased from commercial sources. For construction of dose-response curves, each preparation was exposed to a single concentration of one agent for 30 min.

3.1. Dexmedetomidine and clonidine depress the slow VRP The slow V R P was depressed by dexmedetomidine at concentrations from 0.5-10 nM (fig. 1). The effect appeared within 10-20 rain following initial contact with the drug and continued to develop slowly; the 30-rain period at which measurements of amplitude were made does not represent steady state. The depressant effect of dexmedetomidine was reversible on prolonged (1-2 h) washing with drug-free solution and was stereospecific; the l-isomer of medetomidine, l-medetomidinc, had no effect on the slow V R P at concentrations up to 1 p,M (fig. 1). Exposure to high concentrations of l-medetomidine, however, antagonized the depressant effect of low concentrations of dexmedetomidine. Figure 2 shows a logarithmic dose-response curve for depression of the slow V R P by dexmedetomidine and clonidine. Dexmedetomidine produced a very marked reduction in amplitude of the slow VRP, but usually did not completely abolish the response; at 10 nM and above, average depression at a point 3 s after the stimulus was 80% (fig. 2). Clonidine is the prototypical a2-adrenoceptor agonist. It was of interest to examine the sensitivity of the slow V R P to clonidine in order to test whether the relative sensitivities to dexmedetomidine and clonidine correspond to analgesic potency ratios between the two agents demonstrated in vivo. The slow V R P was reversibly depressed by clonidine at concentrations 20 nM and above (fig. 2). Clonidine was maximally effective at approximately 200 nM. Higher

C

D5nM

W

0.05 mV '

C

LllaM

Fig. 1. The slow ventral root potential (slow VRP) is reversibly and stereospecifically depressed by dexmedetomidine (D). A single stimulus to a lumbar dorsal root ew)kes fast mono- and polysynaptic reflexes in the ventral root (lumped with the stimulus artifact as the spike-like upward deflection early in each record), followed by the slow VRP; note the 2 s time calibration mark. Top row. Control (C): D (5 nM) applied for 30 min depresses the slow VRP; recovery on washing (W) follows perfusion with drug-free solution, in this case for 2 h. Bottom row. The l-isomer of medetomidine (L) 1 aM has no effect after 30 min. Records are averages of 5 sweeps, filtered once through a digital filter with a 20 ms time constant.

295 DEpression (Z)

1O0

versal of dexmedetomidine depression was usually apparent within 15 min after antagonist application and, once initiated, proceeded rapidly, often developing from detectable to complete within the 250 s sampling period required to collect data for the averaged response. These antagonists themselves had little effect on the slow V R P (fig. 3).

• Dmed

o lion

8O

/I

40

3.3. Characterization of the adrenoceptor 2O

o° 10 -I

j lO o

lO l

10 2

10 3

10 4

C o n c e n t r o t 1on (nH)

Fig. 2. Dexmedetomidine (Dmed) and clonidine (Clon) depress the slow VRP. Amplitude of the slow VRP was measured from baseline at 3 s after the stimulus (see fig. 1). Depression was measured as the difference between control and drug-treated amplitude 30 rain after the beginning of drug application and calculated as a percentage of the control response (vertical axis). Dined depression is linearly related to logarithmic concentration; clonidine has biphasic effects. Clonidine concentrations above 200 nM are less effective in depressing the slow VRP, and time-dependent biphasic effects can be observed within a single experiment. Data points are means of 3-15 individual preparations, each preparation exposed to a single drug concentration; error bars are S.E.M.

concentrations of clonidine were less effective. Within individual experiments a time-dependent biphasic effect was often seen, an initial depression following clonidine application followed by a partial recovery toward control levels. Maximal depression of the slow V R P was approximately 80% for both dexmedetomidine and clonidine. The concentrations (ECs0) associated with half-maximal depression of 40% were 0.5 nM and 50 nM, giving a dexmedetomidine : clonidine potency ratio on this basis of 100: 1. If maximally effective concentrations are compared, the potency ratio is approximately 20 : 1.

A series of adrenoceptor agonists and antagonists (table 1) was probed for ability to depress the slow V R P or to interact with dexmedetomidine depression. Phenylephrine, an cq-adrenoceptor agonist, had no effect on the slow V R P at concentrations up to 1 p.M (N = 4). At higher concentrations (20 and 50 /.tM) phenylephrine was associated with apparently excitatory effects manifested as rhythmic ' b u r s t i n g ' discharges superimposed on the slow VRP. Norepinephrine itself (100 nM-1 ~ M ) displayed time-dependent biphasic behavior somewhat similar to that observed with clonidine but more pronounced; initial depression was followed by complete recovery to control values after 30 min exposure (N = 6). The eq-adrenoceptor antagonist prazosin (100 nM) was ineffective in antagonizing dexmedetomidine depression (N = 4). In single experiments the /3-adrenoceptor agonist isoproterenol (10 /tM) had no effect and the /3-adrenoceptor antagonist propranolol ( 1 0 / z M ) did not antagonize dexmedetomidine depression. Attempts were made to distinguish between ef2A- and a2B-adrenoceptor subtypes on the basis of sensitivity to

0.06 m_V

C

D 1 nM

D 1 nM R 5 0 n M

C

D2nM

D2nM

3.2. a-A drenoceptor antagonists The alkaloid rauwolscine and the imidazole atipamezole (Virtanen et al., 1989), two adrenoceptor antagonists with a high degree of selectivity for the a2-adrenoce ptor, were tested for ability to reverse the depressant effect of dexmedetomidine. Dexmedetomidine was applied for 30 min as usual and the effect noted. The preparation was then exposed to the same concentration of d e x m e d e t o m i d i n e c o m b i n e d with one of the antagonists. U n d e r these conditions both rauwolscine and atipamezole readily antagonized depression of the slow V R P induced by dexmedetomidine (fig. 3). Effective rauwolscine antagonism was observed beginning at concentrations 10 times the applied dexmedetomidine concentration, i.e., 50 nM rauwolscine could partially antagonize the effects of 5 nM dexmedetomidine. Re-

A200nM

0.06 mV i

C

R 100 nM

Fig. 3. a2-Adrenoceptor antagonists reverse dexmedetomidine's action on the slow VRP. Top row. Control (C); dexmedetonudine (D) l nM is antagonized by rauwolscine (R) 50 nM; second row, the effect of dexmedetomidine 2 nM is reversed by atipamezole (A) 200 nM. Bottom row. Effective antagonist concentrations of rauwolscine (100 nM) have little effect of their own.

296 TABLE 1 Pharmacology of the slow VRP. The pharmacology of slow VRP depression characterizes the adrenoceptor as a 2, but the subtype could not he identified as either et2A or a2a. a~ (phenylephrine) and ,8 (isoproterenol) adrenoceptor agonists did not depress the slow VRP. The a2-adrenoceptor antagonists rauwolscine and atipamezole antagonized dexmedetomidine depression, as did prazosin at moderate to high concentrations. The approximate ratio between equieffective antagonist concentrations of prazosin and rauwolscine (see text) was 100: 1.

Agent

('oncentration

Slow VRP depressed

Dexmedetomidine Clonidine Oxymetazoline Phenylephrine Isoproterenol Norepinephrine

0.5-10 nM 20-200 nM 10 nM-1 .aM 1 p.M 10 ,aM 100 nM-I ,aM

Yes Yes Inconsistent No No Transiently

Agent

Concentration

Antagonized dexmedctomidine depression

50 nM 50 nM 100 nM 500 nM-50 p.M 10 # M 5 `aM 1 .aM

Yes Yes No Yes No No Slight, pretreatment only

A gonists

3. 4. Non-adrenergic intidazole receptors

Antagonists Rauwolscine Atipamezole Prazosin Prazosin Propranolol Cimetidine Naloxone

oxymetazoline, an agonist with some selectivity for %A-receptors, and prazosin, an antagonist displaying more selectivity for azB-receptors than rauwolscine on the basis of binding studies (Bylund et al., 1988). Oxymetazoline produced inconsistent effects ranging from depression to enhancement of the slow VRP in the range 10 nM-1 p,M (N = 6). The monosynaptic reflex was irreversibly abolished by this agent at very low (10 nM) concentrations.

C

D5nM

D5nM

Prazosin effectively antagonized the effects of dexmedetomidine at concentrations above 500 nM (N = 9) (fig. 4). The protocol, which rigorously demonstrates functional antagonism to the effects of the agonist, does not permit calculation of exact pA 2 values. Relative potencies of prazosin and rauwolscine as antagonists were estimated from the minimum concentration which completely reversed the effect of 5 nM dexmedetomidine. This value for rauwolscine is 100 nM; for prazosin, 10 p,M. The ratio of their effectiveness as functional antagonists is therefore approximately 100: 1. However, at this concentration, prazosin alone enhanced the slow VRP (fig. 4), suggesting that this agent has other effects than simply displacing dexmedetomidine.

P 10 laM

Dexmedetomidine and atipamezole, but not rauwolscine, share an imidazole structure (Virtanen, 1989). In other tissues an imidazole binding site distinct from an a2-adrenoceptor has been identified (Ernsberger et al., 1988). In order to test the role of such a receptor in mediating depression of slow VRP, the effect of the imidazole histamine |[2 receptor antagonist cimetidine was examined. Cimetidine (5 p,M) had no effect itself and did not antagonize the depressant effect of dexmedetomidine (5 nM) on the slow VRP, either when applied together with dexmedetomidine in the protocol outlined above for the a2-adrenoceptor antagonists, or applied 30 min before dexmedetomidine. 3.5. Interaction with an opiate receptor

Evidence from other studies demonstrates links between adrenoceptor-mediated ana opiate receptor-mediated analgesia. The slow VRP is sensitive to opiates (Yanagisawa et al., 1985). The opiate antagonist naloxone (1 ~M, a concentration which blocks opiate effects in this preparation) did not antagonize the effects of dexmedetomidine when applied in the antagonist protocol sequence of dexmedetomidine followed by the combination of naloxone and dexmedetomidine (N = 3). However, when the preparation was pretreated with naloxone for 30 min, the effects of dexmedetomidine were attenuated: 2 nM dexmedetomidine was ineffective in the 30 min exposure period (N = 3), and 5 nM produced only' a slight (20~) decrease in VRP amplitude (N = 2). 3.6. Peptide neurotransmission

C

P 10 p.M

Fig. 4. Prazosin at moderate to high concentrations (500 nM and higher) antagonized the effects of dexmedetomidine but also enhanced the slow VRP by itself. Top row. Control (C); reversal of dexmedetomidine (D) (5 nM) effect by 10 a M prazosin (P). Bottom row. Prazosin (10 `aM) itself increases slow VRP amplitude.

The ipsilateral slow VRP has been reported to be sensitive to the substance P antagonist spantide, but less so than the contralateral (Yanigisawa et al., 1982: Otsuka and Yanagisawa, 1988). We verified that spantide (10-20 p,M) considerably inhibited the ipsilateral

297

C

S 16pM

0.2 rnV L

lOs

C

t

D2nM

t

D2nM R 100 nM

Fig. 5. Interaction between dexmedetomidine and substance P. Top row. Control (C); depression of the slow VRP by the substance P antagonist spantide (S) (16 ~M) indicates that this reflex is in part dependent on peptide neurotransmission. Bottom row. Dexmedetomidine (D), at concentrations which depress the slow VRP, also depresses the response to substance P. At the arrows substance P (2 #M) was ejected by a brief (800 ms) pressure pulse to a micropipette whose tip was positioned near the insertion of the dorsal root. Dexmedetomidine (2 nM) depresses the response to substance P; the effect is antagonized by 100 nM rauwolscine (R). slow V R P in these studies (fig. 5). Brief applications of substance P (2-20 p.M) from a micropipette near the insertion of the dorsal root evoked a slow VRP, as has been previously reported (Yanigisawa et al., 1982; Otsuka and Yanagisawa, 1988). Dexmedetomidine (0.510 nM) depressed the substance P-evoked V R P as effectively as it depressed the slow V R P evoked by dorsal root stimulation; this effect of dexmedetomidine was also antagonized by rauwolscine (fig. 5).

3. Z The monosynaptic reflex The m o n o s y n a p t i c reflex was depressed by dexmedetomidine at concentrations (50-500 nM) approxi-

I

I

C

0.5mY s

D 500 nM

W

'!1 o2rv C

L10gM

Fig. 6. High concentrations of dexmedetomidine reversibly and stereospecifically depress the monosynaptic reflex. Top row. Control (C); dexmedetomidine (D) 500 nM; recovery on washing with drug-free solution (W). Bottom row. The l-isomer of medetomidine (L) 10 /*M shows no effect. Although the response to dexmedetomidine is stereospecific, it is not antagonized by any of the antagonists listed in table 1. Records are averages of five sweeps.

mately 10 times higher than those which depressed the slow V R P (fig. 6). Depression of the m o n o s y n a p t i c reflex was reversible and stereospecific; concentrations of l-medetomidine up to 10 p,M had no effect (fig. 6). Unlike dexmedetomidine, clonidine ( 1 0 / , M ) did not affect the m o n o s y n a p t i c reflex. N o n e of the antagonists tested (table 1) antagonized dexmedetomidine's effect on this response. In particular, the ~2-adrenoceptor antagonists rauwolscine and atipamezole did not antagonize dexmedetomidine's effect even at very high concentrations, at which they themselves exerted depressant effects on the m o n o s y n a p t i c reflex.

4. Discussion Dexmedetomidine reversibly depressed the slow V R P in neonatal rat spinal cord. The pharmacological profile of agonist and antagonist effectiveness characterizes the receptor(s) responsible as a2-adrenoceptors. It was not possible to identify the receptor as either ~2,, or ~2BThe effects of oxymetazoline, an ~ : - a d r e n o c e p t o r agonist with some selectivity for the Ot2A-SUbtype (Bylund et al., 1988), were inconsistent and contaminated by irreversible depression of the m o n o s y n aptic reflex. Prazosin, which binds more strongly to a2u than to ~2A-receptors (Bylund et al., 1988), did antagonize the effects of dexmedetomidine at moderate concentrations. The conditions and assumptions necessary for calculating pA 2 values are not satisfied in the protocol of these experiments, since neither agonist nor antagonist was at equilibrium. However, assuming no pharmacokinetic differences between the two antagonists, the effective equivalent functional antagonist concentrations of rauwolscine (100 nM) and prazosin (10 txM) show a ratio of approximately 1:100. This is between their affinity ratios for the ~2u-receptor subtype (10: 1) and the O~2A-SUbtype (1000: 1) (Bylund et al., 1988). However, the effects of prazosin itself at these concentrations make it difficult to c o m p a r e the present studies, which examine function, with studies which examine only binding affinities. The neonatal rat spinal cord requires validation as a relevant preparation for the study of nociception and analgesia. Connections are not yet mature (Fitzgerald et al., 1987; Fitzgerald. 1985), and receptor populations may be different from the adult. However, a n u m b e r of correspondences to the adult situation emerged from the present study. D e x m e d e t o m i d i n e ' s potency in depressing the slow V R P (0.5-10 nM) is close to that predicted by binding studies (Virtanen, 1989). The stereospecificity and the antagonist effectiveness corresponding to an a2-receptor are similar to the p h a r m a c o logic characteristics of dexmedetomidine's sedative (Doze et al., 1988) and analgesic (Virtanen, 1989) ac-

298 tions in vivo. Of particular interest, the potency ratio between dexmedetomidine and cionidine (between 20 : 1 and 100 : 1, depending on the measure) is similar to the potency ratio of the two agents applied i.t. in the adult rat using tail flick latency as a measure of antinociception (T. Yaksh, personal communication). The approximately linear dose-response relationship of dexmedetomidine, contrasted to the biphasic behavior of clonidine, also corresponds to adult in vivo observations. Clonidine is well known for its decreasing effectiveness as an analgesic at higher doses (Mastrianni et al., 1989). The reason for the difference between the two agents may be either that clonidine is a partial agonist at the a2-receptor, whereas dexmedetomidine is a full agonist; or that clonidine may exert antagonistic cq-adrenoceptor-mediated effects at higher doscs, whereas dexmedetomidine is more selective for the a , receptor. Adrenergic sedation and antinociception are complex phenomena, involving both spinal and supraspinal sites, particularly the locus coeruleus (Aghajanian and Wang, 1987) and other midbrain and medullary areas. The present study cannot eliminate the possibility of supraspinal effects in vivo. However, this study in the isolated spinal cord excludes supraspinal effects, although dexmedetomidine and clonidine may be acting on adrenoceptors normally subject to descending input in the intact nervous system. The sensitivity of the slow VRP to dexmedetomidine suggests a direct spinal action of this agent in analgesia. The location of the a2-adrenoceptor(s) responsible for depression of the slow VRP is not known. The two candidate sites for which there is evidence are the presynaptic terminals of nociceptive afferents and postsynaptic sites on nociceptive interneurons. Substance P release from chick dorsal root ganglion cells is inhibited by norepinephrine as well as by opiates and by other neurotransmitters which act through intermediary G~ proteins; norepinephrine depression of substance P release is antagonized by yohimbine (Holz et al., 1989). Dexmedetomidine does reduce substance P release from adult rat spinal cord (T. Yaksh, pers. comm.) On the postsynaptic side, norepinephrine acting on a2-adrenoceptors directly inhibits nociceptive interneurons in thc substantia gelatinosa (North and Yoshimura, 1984). The comparable effectiveness of dexmedetomidine in depressing the slow VRP evoked either by dorsal root stimulation or by direct application of substance P suggests a postsynaptic site of action should also be considered important for a2-adrenoceptor-mediated actions in spinal cord. It is possible that both pre- and postsynaptic receptors are involved in depression of this nociceptive response. The origin of the slow VRP itself is not firmly established, nor is its correspondence to cellular or reflex responses from more mature animals. Presumably

it is largely generated by motor neurons, although the recording electrode, closely apposed to the cord surface, could detect activity from other cells in the ventrolateral quadrant as well. Evidence that the slow VRP is related to nociception is presented in the introduction. The slow time course is intrinsic to the response rather than the result of multiple synaptic delays: both afferent input (Yoshimura and Jessell, 1989) and substance P applied directly to single spinal interneurons (Murase et al., 1989) elicit responses of very slow time course. The question of interaction with other receptors is an interesting one. "l'here is a rather weak but clear-cut attenuation of the dexmedetomidine effect following naloxone pretreatment. At both peripheral and central nervous system sites, opiate and o~2-adrenoceptors operate on the same ion channels, but the receptors are distinct (Aghajanian and Wang, 1987; Holz et al., 1989). In vivo. naloxone pretreatment partially blocks adrenergic analgesia (Loomis et al., 1987). There is extensive evidence for cross-tolerance and potentiation between opiate and adrenergic analgesic agents. It has been proposed that o~-adrenergic analgesics act in part by releasing endogenous opiates. Alternatively, it may be that endogenous opiate activity serves to prime common effector machinery or to otherwise sensitize neurons to a~-adrenoceptor-mediated effects. With respect to other neurohumoral agents, histamine H2 receptors are involved in anti-analgesic effects at other sites (Gogas and Hough, 1989; Gogas et al., 1989). Cimetidine's ineffectiveness in the present study, either alone or as an antagonist to dexmedetomidine, suggests that neither an U 2 receptor nor another non-adrenergic imidazole receptor is involved in dexmedetomidine's actions or in generation of the slow VRP. However, since cimetidinc appears to differ from other U 2 antagonists (Gogas et al., 1989), further tests of this possibility might be rewarding. The monosynaptic reflex was also depressed by dexmedetomidine but not by clonidine at concentrations much higher than those which maximally depressed the slow VRP. Although the effect was stereospecific for the d-isomer, it was not antagonized by selective antagonists for o~2-adrenoceptors: the antagonists themselves depressed the monosynaptic reflex at high concentrations. The receptors responsible for dexmedetomidine depression of the monosynaptic reflex thus seem to be different from those which mediate depression of the slow VRP. Neither the a~-adrenoceptor antagonist prazosin nor cimetidine antagonized dexmedetomidine's effect on the monosynaptic reflex, even at very high concentrations. The receptors thus appear not to be readily identifiable as 0~-adrenergic. They may belong to a class of receptors, functionally as yet poorly characterized, which are sensitive to some a2-adrenoceptor agonists but distinct from the o~2-adrenoceptor (Michel et al., 1989).

299

In conclusion, the relatively new a2-adrenoceptor agonist dexmedetomidine potently depressed a nociceptive spinal response in vitro, possibly in part by a postsynaptic inhibition of peptide-mediated neurotransmission. The pharmacologic characteristics of its action closely parallel observations in vivo. The results suggest that the analgesic effect of systemically applied dexmedetomidine has an important spinal component, and that the isolated neonatal spinal cord is a valid preparation for further exploration of the analgesic properties of this and other agents.

Acknowledgements Supported by N I H Grant NS13108 to JJK, by GM30232 to MM, and by Farmos Group Ltd., Turku, Finland. We are grateful to A. Tarasiuk at the Unit of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, for teaching the preparation to our laboratory.

References Aghajanian, G.K. and Y.-Y. Wang, 1987, C o m m o n alpha 2- and opiate effector mechanisms in the locus coeruleus: intracelhilar studies in brain slices, Neuropharmacology 26, 792. Akagi, H., S. Konishi, M. Otsuka and M. Yanagisawa, 1985, The role of substance P as a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord, Br. J. Pharmacol. 84, 663. Bhx)r, B.C. and W.E. Flacke, 1982, Reduction in halothane anesthetic requirement by clonidine, an alpha-adrenergic agonist, Anesth. Analg. 61, 741. Bylund, D.B., C. Ray-Prenger and T.J. Murphy, 1988, Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype, J. Pharmacol. Exp. Ther. 245, 600. Chance. W.T., 1986, The role of brain and spinal cord norepinephrine in autoanalgesia, Ann. N.Y. Acad. Sci. 467, 309. Coombs, D.W., R.L. Saunders, J.D. Fratkin, L.E. Jensen and C.A Murphy, 1986, Continuous intrathecal hydromorphone and clonidine for intractable cancer pain, J. Neurosurg. 64, 890. Doze, V.A., B.-X. Chen, Z. Li and M. Maze, 1988, Pharmacologic characterization of the receptor mediating the hypnotic action of D-medetomidine, Acta Vet. Scand. Suppl. 85, 61. Eisenach, J.C., D.M. Dewan, J.C. Rose and J.M. Angelo, 1987, Fpidural clonidine produces antinociception, but not hypotension, in sheep, Anesthesiology 66, 496. Engelman, E., M. Lipszyc, E. Gilbart, P. Van der Linden, B. Bellens, A. Van Romphey and M. De Rood, 1989, Effects of clonidine on anesthetic drug requirements and hemodynamic response during aortic surgery, Anesthesiology 71, 178. Ernsberger, P., R. Giuliano, R.N. Willette, A.R. Granata and D.J. Reis, 1988. Hypotensive action of clonidine analogues correlates with binding affinity at imidazole and not alpha-2-adrenergic receptors in the rostral ventrolateral medulla, J. Hypertens. Suppl. 6, 5554. Fitzgerald, M., 1985. The post-natal development of cutaneous afferent fibre input and receptive field organization in the rat dorsal horn, J. Physiol. 364, 1. Fitzgerald, M., A.E. King, S.W.N. T h o m p s o n and C.J. Woolf, 1987,

The postnatal development of the ventral root reflex in the rat; a comparative in vivo and in vitro study, Neurosci. Lett. 78, 41. Flacke, J.W., B.C. Bloor, W.E. Flacke, D. Wong, S. Dazza, S.W. Stead and H. Laks, 1987, Reduced narcotic requirement by clonidine with improved hemodynamic and adrenergic stability in patients undergoing coronary bypass surgery, Anesthesiology 67, 11. Garcia-Arraras. J.E., T. Murakoshi, M. Yanagisawa and M. Otsuka, 1986, Descending inhibition of slow spinal reflex in an in vitro preparation of the newborn rat and its possible involvement in pain control, Brain Res. 379, 188. Ghignone, M.. O. Calvillo and L. Quintin, 1987, Anesthesia and hypertension: the effect of clonidine on perioperative hemodynamics and isoflurane requirements, Anesthesiology 67, 3. Gogas, K.R. and L.B. Hough, 1989, Inhibition of naloxone-resistant antinociception by centrally administered 1"12-antagonists, J. Pharmacol. Exp. Ther. 248, 262. Gogas, K.R., L.B. Hough, N.B. Eberle, R.A. Lyon, S.D. Glick, S.J. Ward, R.C. Young and M.E. Parsons, 1989, A role for histamine and Hz-receptors in opioid antinociception, J. Pharmacol. Exp. Ther. 250, 476. Goldstein, J.A., 1983, Clonidine as analgesic, Biol. Psychiat. 18. 1339. Gordh, T.E. and A. Tamsen, 1983, A study on the analgesic effect of clonidine in man, Acta Anaesth. Stand. 78. 72. Holz, G.G., R.M. Kream, A. Spiegel and K. Dunlap, 1989, G proteins couple alpha-adrenergic and G A B A B receptors to inhibition of peptide secretion from peripheral sensory neurons, J. Neurosci. 9, 657. Kaukinen, S. and K. Pykko, 1979, The potentiation of halothane anaesthesia by clonidine, Acta Anaesth. Scand. 23, 107. l,c~mis, C.W., K. Jhamandas, B. Milne and F. Cervenko, 1987, Monoamine and opioid interactions in spinal analgesia and tolerance, Pharmacol. Biochem. Behav. 26. 445. Mastrianni, J.A., F.V. Abbott and G. Kunos, 1989, Activation of central mu-opioid receptors is involved in clonidine analgesia in rats, Brain Res. 479, 283. Michel. M.C., O.-t-. Brodde. B. Schnepel, J. Behrendt. R. Tschada, H.J. Motulsky and P.A. Insel, 1989, [31t]Idazoxan and ,some other alpha-2-adrenergic drugs also bind with high affinity to a nonadrenergic site, Mol. Pharmacol. 35, 324. Murase, K., P.D. Ryu and M. Randic, 1989, Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett. 103, 56. North, R.A. and M. Yoshimura, 1984. The actions of noradrenaline on neurones of the rat substantia gelatinosa in vitro, J. Physiol. 349, 43. Otsuka, M. and S. Konishi, 1974, Electrophysiology of m a m m a l i a n spinal cord in vitro, Nature 252, 733. Otsuka, M. and M. Yanagisawa, 1988, Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat, J. Physiol. 395. 255. Reddy, S.V.R., J.L. Maderdrut and T.L. Yaksh, 1980, Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther. 213, 525. Segal, I.S., R.G. Vickery, J.K. Walton, V.A. Doze and M. Maze, 1988, Dexmedetomidine diminishes halothane anesthetic requirements in rats through a postsynaptic alpha2 adrenergic receptor, Anesthesiology 69, 818. Tamsen, A. and T. Gordh, 1984, Fpidural clonidine produces analgesia, Lancet 28, 231. Virtanen, R., 1989, Pharmacological profiles of medetomidine and its antagonist, atipamezole, Acta Vet. Scand. Suppl. 85, 29. Virtanen, R., J.-M. Savola and V. Saano, 1989, Highly selective and specific antagonism of central and peripheral alpha 2-adrenoceptors by atipamezole, Arch. Int. Pharmacodyn. Ther. 297, 190. Yaksh, T.L. and S.V.R. Reddy, 1981, Studies in the primate on the analgetic effects associated with intrathecal actions of opiates. alpha-adrenergic agonists and baclofen, Anesthesiology 54, 451.

300 Yanagisawa, M., T. Murakoshi, S. Tamai and M. Otsuka, 1985, Tail-pinch methcxl in vitro and the effects of some antinociceptive compounds, European J. Pharmacol. 106, 231. Yanigisawa, M., M. Otsuka. S. Konishi, H. Akagi, K. Folkers and S. Rosell, 1982, A substance P antagonist inhibits a slow reflex

response in the spinal cord of the newborn rat, Acta Physiol. ,~and. 116. 109. Yoshimura, M. and T.M. Jessell, 1989, Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro, J. Neurophysiol. 62, 96.