The Journal of Neuroscience, August 1994, 74(E): 4965-4971 Inhibitory Actions of S,-, a,-, and p-Opioid 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, deltorphin, spinal cord slice, EPSP, dorsal root entry zone after blocking inhibitory inputs with 6-opioid receptor, DAMGO, naltriben, 7-benzylidene- bicuculline and strychnine, and NMDA receptors with o-2- naltrexone (BNTX), naloxone] 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 agonists hyperpolarize 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 solutions reduce the inhibitory effectsof opioid oxone competitively antagonized DAMGO as well as DPDPE, receptor agonists on 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 agonists decreaseboth the frequency of miniature EPSPs and demonstration of functional subtypes of the &opioid recep- the amplitude ofevoked EPSPsin dorsal horn neurons in 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]enkephalin (DPDPE) does not 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 neurons in 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 using the 6, receptor antagonist 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).