The Journal of Neuroscience, August 1994, 74(E): 4965-4971

Inhibitory Actions of S,-, a,-, and p- Receptor Agonists on Excitatory Transmission in Lamina II Neurons of Adult Rat Spinal Cord

Steven R. Glaum,’ Richard J. Miller,’ and Donna L. Hammond* Departments of lPharmacoloaical and Phvsioloqical Sciences and ‘Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois 60637 .

This study examined the electrophysiological consequences tor in rat spinal cord and indicate that activation of either of selective activation of 6,-, 6,-, or r-opioid receptors using 6,- or Qopioid receptors inhibits excitatory, glutamatergic whole-cell recordings made from visually identified lamina afferent transmission in the spinal cord. This effect may me- II neurons in thin transverse slices of young adult rat lumbar diate the ability of 6, or 6, receptor agonists to produce an- spinal cord. Excitatory postsynaptic currents (EPSCs) or po- tinociception when administered intrathecally in the rat. tentials (EPSPs) were evoked electrically at the ipsilateral [Key words: DPDPE, , spinal cord slice, EPSP, dorsal root entry zone after blocking inhibitory inputs with 6-, DAMGO, , 7-benzylidene- bicuculline and strychnine, and NMDA receptors with o-2- (BNTX), ] amino+phosphonopentanoic acid. Bath application of the p receptor agonist [D-Ala2, KMePhe4, Gly5-ollenkephalin (DAMGO) or the 6, receptor agonist [D-Pen2, o-PerF]en- The dorsal horn of the spinal cord is an important site for the kephalin (DPDPE) produced a log-linear, concentration-de- production of antinociception by K- and 6-opioid receptor ag- pendent reduction in the amplitude of the evoked EPSP/ onists (Yaksh, 1993). Postsynaptic, as well as presynaptic, sites EPSC. By comparison, the 6, receptor agonist [D-Ala2, of action are thought to mediate this effect. A postsynaptic site Glu4]deltorphin (DELT) was unable to reduce the evoked of action is suggestedby the existence of enkephalinergic syn- EPSP/EPSC by more than 50% at 100 PM, the highest con- apseson dorsal horn neurons (Hunt et al., 1980; Aronin et al., centration tested. At concentrations that reduced evoked 1981; Ruda, 1982; Sumal et al., 1982) and by the ability of EPSP/EPSCs by 40-60%, neither DAMGO, DPDPE, nor DELT opioid receptor agoniststo inhibit the excitation of dorsal horn decreased the amplitude of the postsynaptic current pro- neurons produced by iontophoretic application of excitatory duced by brief pressure ejection of (S)-cu-amino-3-hydroxy- amino acids (Belcher and Ryall, 1978; Zieglgansbergerand Tul- 5-methyl-4-isoxazole-propionic acid, suggesting a presyn- loch, 1979; Willcockson et al., 1984). In addition, w-opioid re- aptic site of action of these opioid receptor agonists. Bath ceptor agonistshyperpolarize and decreasethe input resistance application of 200 nh! naltriben (NTB), a 8, receptor antago- of dorsal horn neurons in slicesof the rat spinal cord or spinal nist, competitively increased the EC,, of DELT by 15.3-fold, trigeminal nucleus (Murase et al., 1982; Jeftinija, 1988; Grudt but did not antagonize either DPDPE or DAMGO. The EC,, and Williams, 1994) primarily by increasing one or more K+ of DELT was further increased by 169.7-fold in the presence conductances (Yoshimura and North, 1983; Grudt and Wil- of 1 PM NTB. However, this high concentration of NTB also liams, 1994). A presynaptic site of action is suggestedby the increased the EC,, of DPDPE by about threefold in a non- ability of opioid receptor agoniststo inhibit the releaseof neu- competitive manner and antagonized DAMGO in a noncom- rotransmitters from primary afferent neurons (Go and Yaksh, petitive manner. In contrast, bath application of 33 or 100 1987; Collin et al., 199 1; Kangrga and RandiC, 1991) and by nM 7-benzylidenenaltrexone (BNTX), a 6, receptor antago- the marked decreasein the number of opioid receptor binding nist, produced a concentration-dependent, noncompetitive sites in the dorsal horn that occurs after dorsal rhizotomy or antagonism of DPDPE, but did not antagonize DELT. A mod- the destruction of small diameter primary afferents by capsaicin est noncompetitive antagonism of DAMGO occurred in the (reviewed by Bessonet al., 1989). In addition, low Ca’+/high presence of 100 nM BNTX. Bath application of 500 nM nal- Mg’+-containing solutionsreduce the inhibitory effectsof opioid oxone competitively antagonized DAMGO as well as DPDPE, receptor agonistson synaptic transmission in the dorsal horn increasing their EC,, values by 13.3- and 2.5-fold, respec- (Murase et al., 1982; Hori et al., 1992). Finally, opioid receptor tively. These results provide the first electrophysiological agonistsdecrease both the frequency of miniature EPSPs and demonstration of functional subtypes of the &opioid recep- the amplitude ofevoked EPSPsin dorsal horn neuronsin slices of rat spinal cord (Hori et al., 1992). Received July 7, 1993; revised Jan. 14, 1994; accepted Feb. 17, 1994. These data provide strong support for opioid modulation of This work was supported by United States Public Health Service Grants DA excitatory afferent synaptic transmissionin the spinal cord, par- 06736 to D.L.H. and DA 02575 and DA 02121 to R.J.M. ticularly by N-opioid receptors. However, the role of &opioid Correspondence should be addressed to Dr. Steven R. Glaum, Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 East receptors is lessclear. The prototypic d-opioid receptor agonist 58th Street, Chicago IL 60637. [D-Pet?, o-Pen5] (DPDPE) doesnot affect the mem- Copyright 0 1994 Society for Neuroscience 0270-6474/94/144965-07$05.00/O brane potential or the amplitude of evoked EPSPsrecorded in 4966 Glaum et al. . Inhibitory Actions of 6- and a-Opioid Agonists

modulation of excitatory afferent synaptic transmission in the control spinal cord. Whole-cell recordings were made of visually iden- tified neuronsin lamina II, a principal termination site for small diameter primary afferent fibers that convey nociceptive infor- mation to the central nervous system (reviewed by Willis and Coggeshall,1991; Light, 1992) in thin transverse slicesof the spinal cord obtained from young adult rats. The ability of the 6, receptor agonist DPDPE (Jiang et al., 1991; Sofuoglu et al., 1991, 1993) the 6, receptor agonist [D-Ala’, Glu4]deltorphin (DELT) (Jiang et al., 1991; Sofuoglu et al., 1991; Stewart and Hammond, 1993) or the p receptor agonist[D-Ala’, N-MePhe4, Glys-ollenkephalin (DAMGO) to inhibit evoked excitatory postsynaptic potentials/currents (EPSP/EPSCs)in theseneurons DAMGO was determined. In addition, the pharmacologic specificity of this inhibition wascharacterized usingthe 6, 7-benzylidenenaltrexone (BNTX) (Portogheseet al., 1992; So- fuoglu et al., 1993) and the 6, receptor antagonist naltriben (NTB) (Sofuoglu et al., 199 1; Stewart and Hammond, 1993). -u Materials and Methods Slice preparation. Transverse slices of the rat spinal cord were prepared as previously described (Bleakman et al., 1992). Briefly, Sprague-Daw- recovery ley rats (Holtzman, Madison, WI) of either sex, aged between 28 and 60 d, were deeply anesthetized with ether and a laminectomy was per- formed that exposed the entire thoracolumbar enlargement and an ad- ditional three to four segments rostrocaudally. The rat was then killed “1 /y”“““” by ether overdose, and the spinal cord and surrounding tissue were immediately removed to a dissecting dish. The spinal cord was freed from the surrounding tissue and 175-225 Frn transverse slices were prepared from the region of the thoracolumbar enlargement with a vibrating microtome. Slices were allowed to recover for at least 1 hr in 32°C artificial cerebrospinal fluid (ACSF), which contained (in mM) NaCl, 126; NaHCO,, 26.2; NaH?PO,, 1; KCI, 3; MgSO,, 1.5; CaC&, 2.5; glucose, 10, and was continuously gassed with 95% OZ, 5% COZ. A single slice was then transferred to the recording chamber (volume of 1 ml), where Figure I. High-resolution trace of monosynaptic EPSCs evoked in a it was continuously perfused (4-6 ml min ‘) at room temperature with lamina II neuron and its inhibition by 0.3 PM DAMGO. The neuron ACSF containing 50 +M o-2-amino-5-phosphonopentanoic acid, 10 PM was voltage clamped at -70 mV and EPSCs were evoked at 0.1 Hz. bicuculline, and 10 PM strychnine. Under these conditions, a complete The averaged (n = 5) trace of the EPSC is shown immediately before exchange ofperfusate occurred in IO-25 sec. Whole-cell recordings were application of DAMGO in the perfusate (control), I5 set after DAMGO made with 2-5 Mq patch electrodes containing in mM: K-gluconate, application (DAMGO), and following a 2 min washout (recovery). The 145; MgCI,, 2; CaCI,, 0.1; HEPES, 5; EGTA, 1.1; K,ATP, 5 (pH 7.2). stimulus artifact has been retouched in the figure. Experiments were typically performed under voltage-clamp conditions in the discontinuous mode of the amplifier at a holding potential of -60 to -80 mV. In discontinuous current-clamp experiments, resting k’,,,was held constant at -60 to -80 mV by direct current injection. superficial dorsal horn neurons in slices of rat spinal cord or Monosynaptic EPSP/EPSCs were evoked by single, subthreshold stimuli spinal trigeminal nucleus (Jeftinija, 1988; Grudt and Williams, (30-120 psec, 3-10 V) delivered at 0.1-0.2 Hz by a bipolar tungsten 1994). Similarly, [D-Pen’, L-Pen5]enkephalin,a close structural electrode placed in the ipsilateral dorsal root entry zone. These EPSP/ analog of DPDPE, does not affect the frequency of miniature EPSCs are denoted generically as EPSCs in the subsequent text. Addition of 10 PM 6,7-dinitroquinoxaline-2,3-dione completely suppressed the EPSPsrecorded in lamina I neurons in slicesof rat spinal cord evoked EPSCs, indicating they were mediated by the actions of gluta- (Hori et al., 1992) leading the authors to conclude that excit- mate on the ol-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid atory synaptic transmission is predominantly modulated by fi (AMPA)/kainate receptor subtype (Schneider and Perl, 1988; Yoshi- receptors(Jeftinija, 1988; Hori et al., 1992). Yet, highly selective muraand Jesse&1990). &opioid receptor agonistsproduce antinociception and inhibit Experimental design. Experiments were performed on 66 visually identified lamina II neurons. A single neuron was examined per slice. the responsesof dorsal horn neurons to noxious stimuli when Each slice was exposed to between one and six applications of DAMGO, administered intrathecally in the rat (Sullivan et al., 1989; DPDPE. or DELT alone or in combination with either NTB. BNTX. Malmberg and Yaksh, 1992; Stewart and Hammond, 1993). or naloxone. The opioid receptor agonists were applied in the perfusate Moreover, recent evidence, much of it obtained from in viva for 15-30 set in the absence or continuous presence of the antagonist, which was also applied in the perfusate. In all cases, slices were washed studies,suggests the existence of at least two subtypes of the for 5-25 min between drug applications to ensure sufficient washout of d-opioid receptor (reviewed by Sofuoglu et al., 1991; Mattia et opioid effects. Desensitization under these conditions was not evident al., 1992; Stewart and Hammond, 1993), both of which are now as responses to reapplication of a given dose of agonist varied by ~5%. implicated in the production of antinociception at the level of The potential interaction of DAMGO, DPDPE, or DELT with post- the spinal cord (Sofuoglu et al., 199 1, 1993; Stewart and Ham- synaptic glutamate receptors mediating the evoked EPSCs was studied in six additional neurons. For these studies, 1 mM (S)-AMPA was ap- mond, 1993; but seeMattia et al., 1992). The present study was plied by brief pressure ejection (2-9 psi, IO-50 msec; Bleakman et al., therefore undertaken to reexamine the role of&opioid receptors, 1992) to the soma ofvisually identified laminae II neurons in the absence and in particular the 6, and 6, subtypes of this receptor, in the and presence of equieffective concentrations of DAMGO, DPDPE, or The Journal of Neuroscience, August 1994, 74(8) 4967

- DAMGO

B m DPDPE A - 50 B-vsopA Figure 2. Representative chart re- cordings of evoked EPSCs (regular 1 I c-50 downward deflections) in lamina II neurons and their inhibition by, A, 0.3 UM DAMGO: B. 20 UM DPDPE: or C. w DELT ‘10 PM DELT: These’concentrations of C DAMGO, DPDPE, and DELT reduced the mean EPSC for all neurons to 59.1 + 5.8% (n = 9) 38.4 i- 3.1% (n = 5), and 52.1 f 6.4% (n = 5) of control, respectively. EPSCs were evoked at 0.2 Hz (A) or 0.1 Hz (Band C). The opioid agonists were applied in the perfusate for the duration indicated by the hori- zontal bars.

DELT. Neurons were examined at holding potentials between - 50 and indicating an absenceofdesensitization. Other investigatorshave -70 mV; 0.5 PM tetrodotoxin was added to the pet&sate for these also reported no desensitization to the presynaptic effects of p studies. receptor agonistsin the central nervous system (North, 1993; Statistical analysis. Drug effects were determined by comparing the averaged peak amplitude of five consecutive EPSCs evoked immediately Rhim et al., 1993). Application of a higher concentration of prior to drug application (I,,,,,, ) to the averaged peak amplitude of five DAMGO (3.0 PM) produced an outward current of ~25 pA in consecutive EPSCs evoked at the apparent peak ofthe response (I treatment) . 4 of 10 cells. This current was absent or sharply reduced after I lrPIlmrn#._ . was typically determined 20-40 set after the onset of drug ap- a second application of 3.0 PM DAMGO. The reduction in plication and was expressed as percentage of control: (Z,re.,men,llcon,ro,) x 100. Concentration-effect lines for DELT. DPDPE. or DAMGO were EPSCsproduced by DAMGO was concentration-dependent(Fig. calculated using least squares linear regression analysis of the individual 3A) and a complete suppressionof evoked EPSCs(to O-5% of data and the concentration ofagonist that produced 50% inhibition was control) occurred in 3 of 10 neuronsafter application of 3.0 PM determined. In the case of DAMGO or DPDPE, both of which com- DAMGO; one of these three neurons exhibited an outward pletely suppressed the EPSCs, this criterion corresponded to 50% of control, or the EC,,. In the case of DELT, which suppressed the EPSCs current. The EC,, (95% CL) of DAMGO was 0.5 (0.3-I. 1) FM. PM by only 50% at the highest concentration tested (100 PM), this criterion Application of 0.2-l 00 DPDPE also produced a reversible corresnonded to 75% of control. or the EC,,. Fieller’s theorem was used reduction in EPSCs(Fig. 2B). The duration of the reduction in to determine 95% confidence limits (CL)-of the EC,, or EC,, values EPSCs produced by DPDPE often persisted longer (up to 20 (Finney, 1964). The concentration-effect lines of each agonist in the min) than the inhibition produced by an equieffective concen- absence and presence of the antagonist were compared for parallelism as described by Tallarida and Murray (1987). Where the lines were tration of DAMGO (2-4 min). The reduction in EPSCswas not parallel, an analysis of covariance was used to determine the statistical accompanied by any appreciable changesin holding current significance of differences in the EC,, or EC,, values of the agonist (Zar, (defined as >5 PA) even at 100 FM DPDPE, the highest con- 1974). centration tested. Moreover, no shift occurred in the whole-cell Drugs. DAMGO, DPDPE, DELT, naloxone, bicuculline, D-2-amino- current-voltage relationship determined in three neuronsat this 5-phosphonopentanoic acid, tetrodotoxin, and strychnine were pur- chased from Sigma Chemical Co. (St. Louis, MO). NTB was synthesized concentration. Like DAMGO, DPDPE produced a concentra- by Mr. Peter Yonan of G. D. Searle and Co. (Skokie, IL). BNTX was tion-dependent reduction in EPSCsand was able to completely purchased from Research Biochemicals, Inc. (Natick, MA). (S)-AMPA suppressEPSCs in two of four neurons (to 0% of control) when was purchased from Tocris Neuramin (Bristol, England). tested at 100 FM (Fig. 3B). Its EC,,, (95% CL) was 5.7 (3.7-9.2) PM. Results Application of 0.05-100 FM DELT also reversibly reduced p- and b- reversib1.vinhibit excitatory transmission EPSCsin a concentration-dependent manner. As with DPDPE, Addition of 0.003-3.0 FM DAMGO to the perfusate produced this reduction also often persisted much longer than the inhi- a reversible reduction in monosynaptic EPSCs (Fig. 1). The bition produced by an equieffective concentration of DAMGO reduction in EPSCswas qualitatively graded and persisted for (Fig. 2C). However, the reduction in EPSCsproduced by DELT 2-4 min after the application of DAMGO was terminated (Fig. reached an asymptote of 50% of control at 10 FM. No further 2A). At concentrations 50.3 FM DAMGO, the reduction in reduction was obtained even in the presenceof 100 PM DELT EPSCswas not associatedwith a detectable changein the hold- (Fig. 3C). Thus, unlike either DAMGO or DPDPE, DELT was ing current, suggestingthat it was mediated at a presynaptic unable to completely suppressevoked EPSCs; the maximum locus. Moreover, repeated application of DAMGO at concen- inhibition observed in any one neuron was to 3 1% of control. trations 50.3 FM produced reproducible reductions in EPSCs, The reduction in EPSCs was not accompanied by any appre- 4966 Glaum et al. * Inhibitory Actions of 6- and p-Opioid Agonists

100 A defined in an ancillary seriesof experiments that examined the 1 effects of these opioid receptor agonists on the postsynaptic current evoked by brief pressureejection of (S)-AMPA. As il- lustrated in Figure 4, concentrations of DAMGO, DPDPE, or DELT that were able to suppressevoked EPSCsto 40-60% of control produced no change in the amplitude of the inward current produced by pressureejection of (S)-AMPA. Thus, in the presenceof 1 FM DAMGO, 2 FM DPDPE, or 1 PM DELT the amplitude ofthe AMPA-mediated current was 102.2 + 7.1% (n = 5) 101.3 f 3.3% (n = 6), and 101.1 * 3.1% (n = 5) of control, respectively.

&ol I 0.01 0.1 10 loo effect of NTB on opioid inhibition qf EPSCs DAM&M) Bath application of 200 nM NTB selectively antagonized DELT, 100 6 but not DAMGO or DPDPE (Fig. 3). In the presenceof 200 1 mm nM NTB, the concentration-effect relationship of DELT was ” 60 shifted to the right in a parallel manner and its EC,, (95% CL) was increasedby 15.3-fold to 4.6 (3.0-8.4) FM (Fig. 3C, P < Ll 0.0 1). By comparison,the EC,, (95%CL) for DAMGO or DPDPE 0 in the presenceof 200 nM NTB did not differ from that deter- E. mined for each agonist alone [DAMGO: 0.3 (0.2-0.4) WM; o* DPDPE: 5.0 (2.7-12.7) PM]. A higher concentration of NTB, 1 zp FM, shifted the concentration-effect relationship of DELT fur- w 20 ther right in a parallel manner and increasedthe EC,, (95% CL) of DELT by 169.7-fold to 50.9 (32.9-92.9) PM (Fig. 3C; P < 0.0 1). This high concentration of NTB also produced a modest 09001 0.01 (threefold) rightward shift in the concentration-effect relation- ship of DPDPE (estimated EC,, 18 HM) that was accompanied loo- L by a marked decreasein the maximum effect produced by o-g- C DPDPE, suggestiveof noncompetitive antagonism. A similar “\, ‘+ 60. noncompetitive antagonism was observed with DAMGO. Al- I 4 though 1 FM NTB did not antagonize the inhibition of EPSCs \. \ \ l - produced by 0.03 or 0.3 FM DAMGO, it did antagonize the 60. 01, I inhibition produced by 3.0 HM DAMGO. Application of 1 FM NTB, the highest concentration tested, was by itself without effect and did not either increase or decreasethe amplitude of the evoked EPSCs.

Ejtict of BNTX on opioid inhibition of EPSCs 1 09001 0.01 0.1 1 10 loo Bath application of 33 nM BNTX produced a sixfold rightward DELT (CIM) shift in the concentration-effect relationship of DPDPE. The estimated EC,,, (95% CL) for DPDPE was increasedto 33.7 Figure 3. Concentration-effect relationships of, A, DAMGO; B, (11.3-345) pM. However, this shift was also accompanied by a DPDPE; and C, DELT in the absence and presence of NTB, a fi2receptor marked reduction in the maximal effect produced by the two antagonist. Ordinate, evoked EPSC or manually voltage-clamped EPSP highest dosesof DPDPE, indicative of noncompetitive antag- expressed as percentage ofcontrol (see Materials and Methods); abscissa, onism (Fig. 5B). Increasing the concentration of BNTX to 100 concentration of agonist (wM). Open circles, agonist alone; solid circles, agonist in the presence of 200 nM NTB; solid squares, agonist in the nM resulted in a greater antagonism of DPDPE. Although an presence of I PM NTB. Concentration-effect relationships were obtained EC,,, value for DPDPE could not be calculated, it appearsto be by briefapplication ofthe agonist during continuous application ofNTB. increasedby at least 17-fold (EC,, > 100 FM). By comparison, Symbols indicate the mean f SEM. Where the SEM is not visible, it 100 nM BNTX did not antagonize the inhibition of EPSCspro- is encompassed bv the symbol. In order of ascending concentration, the number of neurons tested with DAMGO alone was 3,3,9, and 10; with duced by DELT (Fig. 5C), but rather causeda slight, leftward DAMGO and 200 nM NTB was 5. 5. 5. and 6: with DAMGO and 1 shift in the concentration-effect relationship of this & receptor PM NTB was 3, 4, and 6; for DPDPB alone was 3, 7, 9, 5, and 4; for agonist, decreasingits EC,, (95% CL) to 0.1 (0.0 l-0.2) PM. How- DPDPE-and 200 nM NTB was 5, 7, 5, and 6; for DPDPE and 1 ELM ever, this concentration of BNTX did appear to noncompeti- NTB was 5, 4, 6, and 5; for DELT alone was 6, 7, 6, 5, 9, 3; for DELT tively antagonizeDAMGO (Fig. 5A). Addition of 100 nM BNTX, and 200 nM NTB was 3, 7, 4, 4, and 4; and for DELT and 1 PM NTB was 5, 4, and 4. the highestconcentration tested, by itselfwas without effect and neither increasednor decreasedthe amplitude of the evoked EPSCs. ciable changes in holding current (defined as >5 PA) even at the highestconcentration of DELT tested. The EC,, (95% CL) .E&ct of naloxonc on opioid inhibition of EPSCs of DELT was 0.3 (0.1-0.5) PM. The effects of naloxone, a well-establishedcompetitive opioid The site of action of DAMGO, DPDPE, or DELT was further receptor antagonist with approximately lo-fold selectivity for The Journal of Neuroscience, August 1994, 74(8) 4969

A - DELT

B - DPDPE

Figure 4. The postsynaptic inward current evoked by brief pressure ejec- tion of the glutamate receptor agonist (QAMPA (1 mM, 12-25msec, 4-6 psi) was unaltered in the presence of bath applied DELT (1 FM,A), DPDPE(2 PM, B), or DAMGO (1 FM, C). Currentsin A-Cwere evokedin neuronsin separate slicesin the presenceof 0 5 PM TTX 10 PM bxuculhne, 10 PM strychmne, and 50 PM D-2-amino-5-phosphono- A $‘iz pentanoicacid.V : A 160 mV: 8: 1 min - -60 mV; C, -5O’gV. ’

the p receptor (Smith and Leslie, 1993) were also assessedto agonistson dorsal horn neurons(Murase et al., 1982; Yoshimura establishthat competitive antagonism of DPDPE or DAMGO and North, 1983; Jeftinija, 1988). Thus, it should have been could be detected in this preparation. Bath application of 500 possibleto observe direct effects of either DPDPE or DELT on nM naloxone produced a parallel rightward shift in the concen- membrane current had these occurred. Finally, no desensiti- tration+ffect relationships of DAMGO (Fig. 5A) and DPDPE zation occurred upon repeated application of either 6 receptor (Fig. 5B), a finding consistentwith competitive antagonism.The agonist. Several investigators have noted the occurrenceofrapid EC,,, (95% CL) of DAMGO was increasedby 13.3-fold to 7.1 desensitization to the postsynaptic, but not the presynaptic in- (5.0-10.5) pM, whereas the EC,, of DPDPE was increasedby hibitory effectsof opioid agonists(Jiang and North, 1992; Rhim only 2.5-fold to 14.1 (10.2-20.3) FM in the presenceof naloxone et al., 1993). (P < 0.01; both agonists).The K,, of naloxone at the p- and 6, The present resultsalso provide the first electrophysiological receptorswas estimated to be 40 and 334 nM, respectively. The evidence for the existenceoffunctional subtypesofthe 6 receptor effect of naloxone on DELT was not examined as competitive in the spinal cord of the rat. In this study, DPDPE and DELT antagonism of DELT by NTB was observed. each suppressedevoked EPSCs. However, they differed with respectto the magnitude of this effect. DPDPE produced a con- Discussion centration-dependent reduction in evoked EPSCsand, at 100 These results provide the first evidence that excitatory, gluta- PM, was able to completely suppressthe evoked EPSCs.In con- matergic afferent transmission in lamina II of the spinal cord is trast, the reduction in evoked EPSCsproduced by DELT reached inhibited by &opioid receptors. This conclusion is basedon the an asymptote of 50% of control at 10 PM. Although such effects ability of either DPDPE or DELT to reduce the amplitude of could occur if DELT was a partial agonistat the samereceptor evoked EPSCsrecorded in visually identified lamina II neurons at which DPDPE was a full agonist, this explanation is unlikely under conditions in which inhibitory transmissionwas blocked. as the effects of DPDPE and DELT were differentially antago- Several observations suggestthat this effect is most likely me- nized by NTB and BNTX. These observations indicate that diated by a presynaptic site of action. First, at concentrations selective activation ofeither 6, or 6, receptorsinhibits excitatory, that reduced evoked EPSCs by 40-60%, neither DPDPE nor glutamatergic afferent transmission in the spinal cord. DELT reduced the amplitude of postsynaptic currents produced Previous in vitro and in viro investigations suggestthat NTB by direct pressureejection of (S)-AMPA. Second, no change and BNTX are competitive receptor antagonists(Portoghese et occurred in holding currents even after administration of 100 al., 1991, 1992; Sofuloglu et al., 1991, 1993). The parallel right- ~M’DPDPE or DELT, suggestingthat neither DPDPE nor DELT ward shifts produced by NTB in the concentration-effect rela- exert a direct, postsynaptic inhibitory effect on lamina II neu- tionship of DELT are consistent with this conclusion. Estimates rons. By comparison, although 1.O PM DAMGO did not reduce of the K,, of NTB in this preparation ranged from 5 nM (using the amplitude of (S)-AMPA-mediated postsynaptic currents, 1 WM data) to 14 nM (using 200 nM data). The close agreement bath application of 3.0 KM DAMGO produced outward currents of these independent estimates also supports a conclusion of in 4 of 10 neurons in the present study, in agreement with competitive antagonism (Kenakin, 1993). Nonetheless, there previous studiesthat usedconventional intracellular electrodes did appear to be some loss of efficacy of the highest concentra- and reported a direct postsynaptic inhibitory action ofp receptor tions of DELT in the presenceof NTB, suggestinga modest 4970 Cilaumet al. * Inhibitory Actions of 6- and p-Opioid Agonists

DAMGO in this preparation. Not unexpectedly, high concen- trations of both NTB and BNTX antagonized DAMGO. Both NTB and BNTX exhibit p receptor antagonist activity at con- centrations loo-fold and 4-fold greater, respectively, than their K,> for the 6 receptor (Portoghese et al., 1991, 1992). Finally, although both BNTX and NTB interact competitively with the 6 binding site, functional characterization of the antagonist ac- tivities of these drugs at F- and &opioid receptors has been restricted to examinations of a single, optimally selective con- centration or dose (Portoghese et al., 1991, 1992; Sofuloglu et al., 199 1, 1993). Although theseconcentrations produce parallel shifts in the dose-effect relationshipsof 6- and p-opioid receptor agoniststhat are compatible with competitive antagonism, the 0.001 0.01 0.1 10 100 DAMGO GM) limited selectivity of these drugs and the agonist activity en- countered at higher doses has precluded performance of the 100. Schild analysesnecessary for definitive characterization of the competitive nature of the antagonism. g 60. Previous studiesof the effects of opioid receptor agonistsin b slicesof rat spinal cord concluded that excitatory afferent trans- E o 60- mission was modulated predominantly by the FL-opioidreceptor (Jeftinija, 1988; Hori et al., 1992). However, the ability ofeither ?z. DPDPE or DELT to reduce evoked EPSCsin a concentration- 0 40. dependent, pharmacologically specific manner in the rat spinal cord indicates that 6 receptorsalso modulate excitatory afferent 20. transmission. One possible explanation for the apparent dis- crepancy betweenthese results and thoseofearlier investigations 1 , . .., ,, is the age of the animals. Slices of spinal cord obtained from &01 0.01 0.1 10 loo neonatal or juvenile rats were used in many of the previous DPDPE GM, studies(Murase et al., 1982; Jeftinija, 1988; Hori et al., 1992). 100 C However, the first 3 postnatal weeks is characterized by contin- 1 Q, ued development and maturation of somatosensorypathways in the spinal cord of the rat. For example, C fibers are the last primary afferent fibers to innervate the spinal cord, entering the 8 60. dorsal horn on or about the day ofbirth (Fitzgerald, 1985, 1987), ?!t. and the reflexes subserved by polymodal C-fibers are not fully g 40. developed until nearly 3 weeks of age (Fitzgerald and Gibson, 1984). Furthermore, neurons in lamina II do not complete their differentiation and maturation processesuntil 3 weeks of age (Bicknell and Beal, 1984). Finally, in contrast to w-opioid re- ceptors, d receptors in the brain and spinal cord do not appear 1 ,_,,,,, ...... ,.,,,, ...... m* until after birth and may not reach adult levels until nearly 3 klo1 0.01 0.1 10 loo DELT (jib, weeks of age (Spain et al., 1985; McDowell and Kitchen, 1986; Jameset al., 1990; but seeAttali et al., 1990). Thus, the results Figure 5. Concentration-effectrelationships of, A, DAMGO; B, of previous studies may have been influenced by the use of DPDPE; and C, DELT in the absenceand presenceof BNTX, a 6, neonatal or juvenile rats. receptorantagonist, or naloxone.Ordinate, evokedEPSC or manually voltage-clampedEPSP expressed as percentage of control (seeMaterials In conclusion, these results provide strong evidence that ac- and Methods);abscissa, concentrationof agonist(PM). Open circles, tivation of either 6,- or &-opioid receptors inhibits excitatory, agonist alone: so/id circles, agonist in the presence of 33 nM BNTX; glutamatergic afferent transmissionin the spinal cord of the rat. solid squares. ‘agonistin the presenceof 106 nM BNTX; open triangles, These resultsare also in excellent agreementwith earlier in viva agonistin the presenceof 500 nM naloxone.Concentration-effect re- investigations of spinal 6 receptors in which intrathecal admin- lationshipswere obtainedby brief applicationof the agonistduring continuousapplication of BNTX. Symbolsindicate the meanf SEM. istration of DPDPE or DELT produced antinociception that Wherethe SEM isnot visible,it isencompassed by the symbol.In order was differentially antagonized by NTB and BNTX (Mattia et of ascending concentration, the numbers of neurons tested with DAM- al., 1992; Sofuoglu et al., 1991, 1993; Stewart and Hammond, GO and,100 tn.4BNTX were 7, 5, and 5; with DAMGO and 500 nM 1993). Thus, inhibition of excitatory, glutamatergic afferent naloxonewere 5, 5, 5, and 5, with DPDPEand 33 nM BNTX were4, transmission in the dorsal horn may mediate the ability of in- 4, 4, and 5; with DPDPEand 100nM BNTX were5, 5, 5, and 5; with DPDPEand 500nM naloxonewere 5.6, 5, and 3; and with DELT and trathecally administered 6, or 6, receptor agoniststo produce 100nM BNTX were5, 5, and 5. antinociception. insurmountable antagonism. Our finding of noncompetitive, References rather than competitive antagonism of DPDPE by BNTX is at Aronin N, DetigliaM, Liotta AS, Martin JB (1981) Ultrastructural odds with previous reports, but is strengthened by our finding localization and biochemicalfeatures of immunoreactiveLeu-en- that naloxone competitively antagonized DPDPE as well as kephalin in monkey dorsal horn. J Neurosci 1:561-577. The Journal of Neuroscience, August 1994, 14(8) 4971

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