Dual opioid modulation of the action potential duration of mouse dorsal root ganglion neurons in culture*

Transcription

1 Brain Research, 491 (1989) Elsevier BRE Dual opioid modulation of the action potential duration of mouse dorsal root ganglion neurons in culture* K.-E Shen I and S.M. Crain 1'2 Departments of I Neuroscience and 2physiology~Biophysics and Rose E Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY (U.S.A.) (Accepted 13 December 1988) Key words: Excitatory opioid receptor; Dorsal root ganglion neuron; Action potential duration; Cyclic AMP; Pertussis toxin-sensitive G protein; Membrane Ca z+ conductance; Membrane K conductance Multiple modulatory effects of opioids on the duration of the calcium component of the action potential (APD) of dorsal-root ganglion (DRG) neurons of mouse spinal cord-ganglion explants were studied. The APD of DRG neuron perikarya has been previously shown to be shortened by exposure to high concentrations of opioids (ca /~M) in about 1/2 of the cells tested. The present study demonstrates that in addition to these inhibitory modulatory effects of opioids, lower concentrations (1-10 nm) of ~-,/~, and K-opioid agonists elicit excitatory modulatory effects, i.e. prolongation of the APD, in about 2/3 of the sensory neurons tested. APD prolongation as well as shortening elicited by 6,/~, and K agonists were prevented by coperfusion with the opioid antagonists, naloxone or diprenorphine (10 nm). APD prolongation induced by the 6-agonist [D-AlaZ-o-LeuS]enkephalin (DADLE) was prevented in the presence of multiple K + channel blockers, whereas excitatory modulation by the specific K-agonist, U-50,488H was not attenuated under these conditions. After treatment of DRG neurons with pertussis toxin (1/~g/ml for several days) or forskolin (50/~M for >15 min), a much smaller fraction of cells showed opioid-induced APD shortening; moreover, a much larger fraction of cells showed opioid-induced APD prolongation, even when tested with high concentrations of DADLE (1-10/~M). These data indicate that opioid-induced APD prolongation is not mediated by pertussis toxin-sensitive G proteins (which have been shown to regulate opioid inhibitory effects) and suggest that elevation of cyclic AMP levels may enhance opioid excitatory responsiveness. Furthermore, our analyses indicate that,u-, 6- and K-subtypes of excitatory as well as inhibitory opioid receptors may be expressed on the same DRG neuron perikaryon under in vitro conditions. If dual opioid modulation of the APD of DRG perikarya also occurs in central DRG terminals this may play a significant role both in nociceptive signal transmission as well as tolerance to opioid analgesia. INTRODUCTION Opioids generally produce inhibitory modulation of nerve cells either by membrane hyperpolarization that decreases the firing rate of action potentials (e.g. in locus coeruleus neurons a6) or by shortening the action potential duration (APD) (e.g. in dorsal root ganglion (DRG) neurons of the mouse 6'65-68 and chick45). These and related opioid inhibitory effects are blocked by pertussis toxin (PTX) which uncouples various types of opioid and other modulatory receptors from regulatory G proteins (e.g. Gi and GO) 1'2'13'29'34'43. Opioid inhibitory modulation of the APD of DRG perikarya is mediated by: (1)/~- and 6-0pioid receptors that increase voltage- and/or calcium-dependent K -conductances, and (2) r- opioid (dynorphin) receptors that decrease a voltage-dependent Ca2+-conductance (see also ref. 9). It has been postulated that similar opioid-induced opening of K channels and/or closing of Ca 2 channels in presynaptic DRG terminals may result in shortening of the APD, reduction in Ca 2 influx and decrease in neurotransmitter release 45,46, In addition to these opioid inhibitory effects on the APD of DRG neurons, we have found that specific 6-,/~- and r-opioid agonists can also prolong * Preliminary reports of this study have been published (see refs. 15, 54, 55). Correspondence: S.M. Crain, Dept. of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, U.S.A /89/$ Elsevier Science Publishers B.V. (Biomedical Division)

2 228 the APD especially when applied at low concentrations (1-10 nm), in contrast to the APD shortening generally observed at higher concentrations (ca. 1 /~M). Opioid prolongation of the APD of DRG neurons has not been noted in most previous electrophysiologic studies (see reviews in refs. 21, 46), except for preliminary reports on adult rabbit nodose ganglion cells in situ, by Higashi et al. 3 and fetal mouse DRG neurons in dissociated cell culture, by Werz & Macdonald 64. In nodose ganglion cells, a definite tendency for APD prolongation by low concentrations of morphine (1 nm) was noted, whereas at higher concentrations ( nm) the APD was shortened. These effects of morphine were prevented in the presence of naloxone 3. The present study shows that similar dual opioid excitatory and inhibitory effects occur in DRG as well as in nodose sensory neurons and that APD prolongation can be elicited by low concentrations of specific d-, /,- and,c-opioid agonists as well as by morphine. In addition, we provide evidence that APD prolongation induced by/*/d-opioid agonists is mediated by receptor subtypes that decrease a voltage-sensitive K + conductance and that the excitatory effects of a kappa agonist appear to increase a voltage-sensitive Ca 2+ conductance. Since sensory DRG neuron perikarya are devoid of synaptic inputs, opioid excitatory effects on these cells are clearly direct actions 1~'16, in contrast to opioid excitation of hippocampal and other CNS neurons where the primary effect appears to involve opioid inhibition of non-opioid inhibitory interneurons TM. Analyses of dual opioid modulation of the APD of DRG neurons in vitro may provide valuable insights into mechanisms of antinociception and tolerance to opioid analgesia. MATERIALS AND METHODS Tissue culture Explants were prepared by dissecting transverse sections of spinal cord with attached DRGs from 13-day-old fetal mice (CD-1, Charles River) 6. The DRG-cord explants were grown on collagen-coated coverslips in Maximow slide chambers. The culture medium consisted of 65% Eagle's minimal essential medium, 25% human placental serum, 10% chick embryo extract, 2 mm glutamine and 0.6% glucose. During the first week in vitro the medium was supplemented with nerve growth factor (NGF-7S) at a concentration of about 0.5 /~g/ml to enhance survival and growth of the fetal mouse DRG neurons. Explants were used for electrophysiologic study after 3-5 weeks in vitro. Pertussis toxin (1 /~g/ml) was added to the nutrient medium of some of the cultures, beginning at 3 weeks in vitro, for >4-5 days. Electrophysiology For electrophysiologic study, the culture coverslip was transferred to a small recording chamber containing about 1 ml of Hanks' balanced salt solution (BSS) supplemented with 4 mm Ca 2 and 5 mm Ba 2. Addition of Ba 2+ and high Ca 2+ enhanced inward Ca 2+ current, and Ba 2 suppressed delayed rectifying K + channels 32, thereby prolonging the APD and providing a prominent baseline response for pharmacologic tests 6'45. In some of the tests, 5 mm Ba 2 was replaced by 5 mm tetraethylammonium chloride (TEA) prior to introduction of opioids (as in studies by Werz & Macdonald and Higashi et al.3 ). To prevent Ba 2+ and Ca 2+ precipitation, the usual bicarbonate/phosphate buffers were replaced by 10 mm HEPES (ph 7.3). The recording chamber was placed on the stage of an inverted microscope which allowed neuronal impalement by a recording micropipette during direct visual monitoring. Intracellular recordings were obtained from 1-10 DRG perikarya selected at random within each ganglion. The micropipettes were filled with 3 M KC1 (resistance about MI2) and were connected via a chloridized silver electrode to a neutralized input capacity preamplifier (WPI-M707A). A bridge circuit allowed simultaneous current passage and voltage recording with a single microelectrode. After impalement of a DRG neuron, brief (2 ms) depolarizing current pulses were applied to evoke action potentials (at a frequency of 0.1 Hz). In some of the experiments, 100 ms depolarizing current pulses were used to study repetitive firing properties of the neurons. Oscilloscope traces of action potentials were recorded on photographic film. Some of the tests were carried out in the presence of multiple K channel blockers at concentrations that have been shown to be effective on DRG neurons 45'6s (see also refs. 46, 47): 5 mm

3 229 TEA, 5 mm Ba 2+ and no Ca 2+ in the bathing fluid, and 2 M CsCI in the intracellular pipette. Perfusion of opioids was begun after the AP and the resting potential (RP) of the neuron reached a stable condition during >4 min pretest periods in control Ca, Ba/BSS as well as in the presence of multiple K channel blockers. Tests were generally made on cells with RPs >50 mv and in some cases, a holding current was applied to maintain the baseline membrane potential at about -60 mv. The latter procedure was regularly utilized during experiments with multiple K channel blockers since impalement of DRG neurons with a CsCl-filled micropipette reduced the RP to about 0 mv 68. Opioid-mediated changes in the APD were considered significant if the APD alteration was >10% of the control value and was maintained for the entire test period (3-5 min). The APD was measured as the time between the peak of the AP and the inflection point on the repolarizing phase. Cell input resistance (Rm) was determined by injecting various amplitude hyperpolarizing current pulses and measuring the resulting steady-state voltage change from the resting potential. The slope of the resulting V-I relationship was taken as the input resistance of the cell. The opioid effects were generally reversed after elimination of the ligand by several rinses of the bath with control medium (3-6 ml). Further control data were provided by many cases where perfusion with opioids resulted in no alteration of the APD (Tables I-V; see further technical details in refs. 6, 16). The following drugs (and their sources) were used: [D-Ala,D-Leu]enkephalin (DADLE; Peninsula Labs), Tyr-D-Pen-Gly-Phe-D-Pen (Pen = penicillamine; DPDPE; Peninsula Labs), Tyr-D-Ala-Gly- MePhe-Gly-ol (DAGO; Peninsula Labs), U-50, 488H (Upjohn), naloxone (Endo), forskolin (FSK, Calbiochem), PTX (List), TEA and other salts (Sigma). Diprenorphine was a gift from Dr. E.J. Simon. Stock solutions were generally prepared in distilled water; FSK was dissolved in ethanol (20 mm) and diluted to 50/aM in BSS. (Control tests with 0.5% ethanol showed no significant effects on the APD or its modulation by opioids.) Two types of data were evaluated: continuous and enumerative. For the former, i.e. magnitude of the APD, we used Student's t. For the latter, i.e. numbers of cells showing opioid-induced APD prolongation or shortening, or no effect, we used a )~2 test of contingency. RESULTS Dual effects of DADLE on the APD of DRG neurons The responsiveness of DRG neurons to opioids was analyzed by measuring the alterations in the APD of DRG perikarya a7'47. The opioid tests were routinely made in BSS containing both high Ca 2+ (5 mm) and Ba 2 (5 mm). A total of 74 DRG neurons (from 45 DRG-cord explants) were studied for sensitivity to 1 /am DADLE. Application of DADLE decreased the APD in 49% of the DRG neurons (n = 36; Table I; Figs. 1A 3 vs A1; 4A 3 vs Az), in good agreement with a previous study by Chalazonitis and Crain 6 on neurons in similar DRGcord explants. The magnitude of APD shortening produced by 1/aM DADLE in these DRG neurons ranged from -17 to -81% (mean + S.E.M.: %; n = 36). Opioid shortening of the APD was antagonized by coperfusion with naloxone (3/aM; n = 4), demonstrating mediation by specific opioid receptors. Among the other 38 neurons, 25 cells (34%) showed an increased APD when 1 /am DADLE was applied (Table I; as occurred more generally with lower DADLE concentrations, e.g. Figs. 1A e vs A1; Fig. 2D; see below). The magnitude of DADLE-induced APD prolongation in these DRG neurons ranged from +23% to +230% ( %; n = 24). Prolongation of the APD started within 1-2 min after DADLE perfusion and was maintained for the entire test period (>5 min). In some of the cells where 1/aM DADLE shortened the APD or was ineffective, the APD showed transient prolongation during the first 3-5 min of rinsing with BSS. This suggested that DADLE might be more effective in eliciting APD prolongation at lower concentrations (see below). No significant alteration in the RP (ca mv) of the DRG neurons (generally <5-10%) occurred during most of the opioid-induced APD prolongation or shortening observed in the present study. In cases where larger decreases or increases in RP were seen during long-term impalements of DRG neurons, control tests showed that they were

4 230 A ~ _ A2 A3_ BalSSS DAOLE DADLE lonm lum DIPRENORPHINE (1OHM) PRETREATMENT A4- A5- A6- DAOLE lonm DADLE I.,. B2.. B3~ TEAIBSS DADLE DADLE 1OhM lum -_/ -/ - NALOXONE (1OHM) PRETREATMENT B4_ BS~ B% DADLE 1OhM OADLE lum Fig. 1. DADLE prolongs the Ca 2+ component of the somatic action potential (APD) of DRG neurons at low concentration and shortens APD at higher concentration. Both effects are prevented by the opioid antagonists, diprenorphine or naloxone. Aa: AP generated by a DRG neuron in balanced salt solution containing 5 mm Ca 2+ and 5 mm Ba 2 (Ba/BSS) in response to a brief (2 ms) intracellular depolarizing current (same 2 ms stimulus was used in all subsequent records). Az: APD is prolonged within 5 rain after addition of 10 nm DADLE. A3: APD is shortened within 5 min after increasing DADLE concentration to 1 /~M. A4_6: tests on the same neuron after BSS rinse and pretreatment with diprenorphine. A4: APD is not altered after 5 min in 10 nm diprenorphine. As: APD is only slightly prolonged after coperfusion of 10 nm DADLE and diprenorphine, in contrast to the marked opioid-induced prolongation elicited prior to introduction of the antagonist (cf. A2). A6: APD is unaltered even after addition of 1 #M DADLE, in contrast to the marked shortening elicited in the absence of diprenorphine (cf. A3). B~: AP generated by another DRG neuron in balanced salt solution containing 5 mm Ca z+ and 5 mm TEA (TEA/BSS). Be: APD is prolonged within 5 min after addition of 10 nm DADLE (as in test made in Ba/BSS:A2). B3: APD is shortened within 2 min after perfusion with 1 #M DADLE (as in A3). B4_6: tests on the same neuron after BSS rinse and pretreatment with naloxone. B4: APD is not altered after 5 min in 10 nm naloxone. Bs: APD is unaltered after addition of 10 nm DADLE, in contrast to the opioid-induced prolongation elicited prior to introduction of the antagonist (cf. Be). B6: APD is unaltered even after addition of 1/aM DADLE, in contrast to the shortening elicited in the absence of naloxone (cf. B3). Note: In this and subsequent figures, records are from DRG neurons in DRG-cord explants tested at 3-7 weeks in vitro. Calibration pulse preceding each AP: 10 mv, 2 ms. A depolarizing stimulus current pulse (2 ms) is displayed on upper trace of each record. 'FABLE I Low concentrations of DADLE prolong the APD of many DRG neurons whereas higher concentrations shorten the A PD Tests were made in 5 mm Ca 2~, 5 mm BaE+/BSS on 170 neurons in 92 DRG-cord explants (from 13-day fetal mice) at >3 weeks in vitro. Initial APDs ranged from 3 to 84 ms (11 +_ 1 ms; n = 170) in all test groups. DADLE effect Proportions (%) of DRG neurons tested on A PD OHM* I #M IO#M (n = 54) (n = 74) (n = 42) Shortened Prolonged Not altered * In x 2 tests, P < vs 1 #M or 10ktM DADLE groups. not causally related to the observed opioid-induced APD prolongation or shortening. The R m of the DRG neurons ranged from 22 to 126 Mg2 (mean Mg2; n = 18) and often decreased during exposure to 10 nm DADLE (by 11 to 62%; mean %, in 13 out of 18 cells). However, the decrease in Rr, showed no correlation with opioid- induced APD alterations. Opioid-induced APD shortening was readily re- versed within a few minutes by several successive rinses of the bath with BSS. In contrast, opioid- induced APD prolongation often reversed relatively slowly during periods of 5-15 min after opioid withdrawal, as observed in similar tests on chronic DADLE-treated DRG neurons ~6. A second acute opioid exposure was often effective in prolonging the APD again when applied after a min BSS rinse (21 out of 29 cells). Preferential APD prolongation of DRG neurons exposed to low concentrations of DADLE A much larger proportion of DRG neurons showed APD prolongation in 10 nm DADLE (76%; e.g. Fig. 1A2, B2), and only 9% showed APD shortening (n = 54; Table I). The magnitude of the APD prolongation elicited by 10 nm DADLE ranged from +13% to +400% ( %). Thus, 10 nm DADLE-induced APD prolongation in some DRG neurons was comparable to, or larger than, the percentage increase elicited by 1 ~M DADLE, although the mean increase in APD was smaller, i.e. +47% vs +70%. An additional series of DRG

5 231 A B C D.,,.J BSS.-.../ _.-._i BSS BSS BSS.... DPDPE lonm U-50488H lonm DAGO 10nM DADLE 100nM DPDPE loonm U-50488H lum DAGO 1 um DADLE lum Fig. 2. Specific/~-, & and K-opioid agonists elicit markedly different alterations of the APD in the same DRG neuron. A: APD is slightly prolonged by the &agonist, DPDPE, at l0 nm and markedly prolonged after increasing to 100 nm (5 min test periods). B: after thorough rinsing in BSS, APD is prolonged by the r-agonist, U-50,488H, both at 10 nm (by 5 min) and 1/~M (by 1 min). C: after BSS rinse, APD is shortened by the # agonist, at 1 #M (by 3 min), whereas 10 nm DAGO is ineffective. D: after BSS rinse, 100 nm DADLE prolongs APD whereas 1 pm DADLE shortens it back to baseline level (3 min test periods). Note: Each record in Figs. 2 and 3 consists of 3 superimposed oscilloscope sweeps. BSS includes 5 mm Ca 2 and 5 mm Ba 2+. neurons from the same batches of DRG-cord explants were tested with 10/~M DADLE. Similar to the 1 #M DADLE series (see above), 55% showed APD shortening and 24% showed APD prolongation (n = 42; Table I). These results demonstrate that DADLE-induced prolongation of the APD of DRG neurons depends critically upon exposure to low (nm) concentrations, whereas higher concentrations (1-10 #M) often induce inhibitory effects. To confirm these dose-response effects of DADLE on the APD of DRG neurons, 3 different concentrations of DADLE (10 nm, 100 nm and 1 ~M) were tested sequentially on the same cell. A total of 20 DRG neurons were studied (Table II). In group A, a dual response to different concentrations was obtained (10 cells), where a low DADLE concentration (10 nm) elicited APD prolongation and higher concentrations (100 nm and 1 #M) elicited APD shortening (e.g. Fig. 1A2,3; Be,3). The magnitude of APD shortening was concentrationdependent: % and % of the control value in 100 nm and 1 #M DADLE, respectively. In groups B and C only APD shortening occurred depending on the DADLE concentration (5 cells). In group D, only APD prolongation occurred when tested at all three DADLE concentrations (5 cells). In 3 of these cases, the APD was progressively increased at higher DADLE concentrations (100 nm-1 ~M) (see also similar dose-dependent effects TABLE II Dose-dependence of opioid-induced alterations of APD tested on individual DRG neurons Each of these 20 cells (9 explants) was tested at all 3 concentrations of DADLE. Test conditions in this and subsequent tables are the same as in Table I unless otherwise specified. Group No. of cells Effect of DADLE concentration on APD tested 10 nm O. 1-1 I~M A 10 prolonged shortened B 4 nochange shortened C 1 shortened shortened D 5 prolonged prolonged

6 232 of DPDPE: Fig. 2A). High and low concentrations of DADLE were also effective in eliciting APD shortening and prolongation in DRG neurons when the 5 mmba 2+ in the BSS was replaced by 5 mm TEA. Low concentrations of DADLE (10 nm) elicited APD prolongation in 84% of the DRG neurons in the presence of 5 mm TEA (Fig. 1B 2 vs B1; n = 19), comparable to 76% of the cells tested in 5 mm Ba 2+ as noted above (Table I). Furthermore, high concentrations of DADLE (1 ym) tested on the same group of neurons in TEA/BSS prolonged the APD in only 21% of the cells and shortened the APD in 68% of the cells (Fig. 1B 3 vs B0, consonant with the results obtained in the presence of Ba/BSS (Table I). However, no significant opioid-induced APD prolongation has been observed in tests made in the absence of Ba 2+ or TEA. Effect of specific opioid agonists on APD of DRG rieurorls More specific opioid agonists were investigated at low concentrations (10 nm) to determine if similar opioid-induced APD prolongation occurred when subtypes of opioid receptors were selectively activated. The specific b-receptor agonist, DPDPE 44, was tested on 18 DRG neurons. Fifteen cells (83%) showed APD prolongation (e.g. Fig. 2A) and only 2 (11%) showed APD shortening (Table III). Moreover, the mean value of the APD prolongation in 10 nm DPDPE was % (n = 15), comparable to that observed in 10 nm DADLE. The specific/~-agonist, DAGO 28, was tested on another series of 34 DRG neurons, also at the 10 nm level. Thirty cells (88%) showed APD prolongation and only 2 (6%) showed APD shortening (Table III). The mean value of the APD prolongation in 10 nm DAGO was % (;7 = 28). Furthermore, 22 of the cells showing DAGO-induced APD prolongation were also tested with a higher concentration of DAGO (1/,M). In 18 of these cells (82%) the APD prolongation was reversed to a shortening and only 2 cells continued to show an APD prolongation in 1 #M DAGO. These results show that DAGO can elicit dual modulatory effects as observed with DADLE, i.e. prolongation of the APD at low concentration (10 rim) and reversal to APD shortening at higher concentrations (1 #M). The specific ~:-agonist, U-50,488H 62, was tested on another group of 37 DRG neurons at 1-10 nm levels. Twenty-nine cells (78%) showed APD prolongation (e.g. Fig. 2B) and only 3 (8%) showed APD shortening (Table III). The mean value of the APD prolongation in 1-10 nm U-50,488H was % (n = 29), substantially larger than elicited by DADLE, DAGO or DPDPE. These tests with low (rim) concentrations of specific opioid agonists raise the possibility that APD prolongation of DRG neurons may be mediated by high-affinity, excitatory subtypes of d-, #- and ~c-opioid receptors, in contrast to the loweraffinity inhibitory receptor subtypes that have been shown to mediate APD shortening in these cells. To determine if these putative excitatory 6-, #- and ~c-opioid receptors coexist on the same DRG neurons, tests were made sequentially with different opioid agonists (DAGO, DPDPE, DADLE and U-50,488H) at different concentrations. In one experiment, a DRG neuron showed progressive increases in the APD when tested in 10 nm and 100 nm concentrations of DPDPE (Fig. 2A), but sustained prolongation occurred in 10 nm and 1 /~M TABLE II1 Low concentrations of specific#-, 6- and lc-opioid agonists are effective in eliciting APD prolongation In this and subsequent tables, 1-3 cells were tested per explant. Opioid effect on APD Proportions of DRG neurons tested (%) DA DLE 10 nm DPDPE 10 nm DAGO 10 nm U-50,488H 1 nm (n = 54) (n = 18) (n = 34) (n = 37) Shortened Prolonged Not altered

7 233 U-50,488H (Fig. 2B). The same cell, on the other hand, showed no APD alteration in 10 nm DAGO but APD shortening in 1 /~M DAGO (Fig. 2C). Subsequent exposure to 100 nm DADLE resulted in APD prolongation, whereas APD shortening was elicited by 1/aM DADLE (Fig. 2D). Other DRG neurons showed varied patterns of opioid responsiveness when tested with specific opioids in different sequences, e.g., APD prolongation in 10 nm DAGO, followed by APD shortening in 10 nm U-50,488H and then in 100 nm DADLE, and finally APD prolongation in 100 nm DPDPE. These results suggest that some DRG neurons can possess both excitatory and inhibitory subtypes of 6-, p- and r-opioid receptors on the same perikaryal membrane. Effect of specific antagonists on opioid-induced APD prolongation of DRG neurons Tests with the opioid antagonists, diprenorphine or naloxone, were carried out in two ways. In one paradigm, a DRG neuron was initially tested to determine if its APD would be prolonged by a specific opioid exposure (5 min test); after a recovery period in BSS (ca. 10 min) and a 5 min exposure to the opioid antagonist, the opioid was then copeffused with the antagonist for an additional 5 min test period. Alternatively, a culture was initially pretreated with an opioid antagonist and a series of DRG neurons was then tested sequentially for evidence of opioid-induced APD prolongation in the presence of the antagonist. Control tests were made with 10 nm DADLE or DAGO on DRG neurons in other cultures from the same batch to ensure that about 2/3 of the cells in these ganglia showed characteristic opioid-induced APD prolongation (as in Table III). Neither naloxone nor diprenorphine altered the APD when tested alone at 10 nm concentrations prior to coperfusion with opioids. The first paradigm was applied to 12 DRG neurons (in 12 explants) which showed APD prolongation when exposed to a low concentration of DADLE (10 nm) (e.g. Fig. 1A2). Subsequent exposure of 11 of these cells to a high concentration of DADLE (1/tM) resulted in characteristic APD shortening in 9 (e.g. Fig. 1A3), whereas 2 cells showed an additional APD prolongation. After a min recovery period (in BSS and then in the antagonist, e.g. Fig. 1A4) the opioid was copeffused with 10 nm diprenorphine. Under these conditions, none of the cells showed significant prolongation of the APD in 10 nm DADLE, nor shortening in 1/~M DADLE (Fig. IA5.6). Coperfusion with 10 nm diprenorphine also prevented the APD prolongation elicited by 1 nm U-50,488H in many DRG neurons (7 out of 8 cells, in 5 explants). Additional coperfusion tests with 10 nm naloxone showed that this opioid antagonist could also reliably prevent DAGO-induced APD prolongation (9 out of 9 cells, in 8 explants) as well as DADLE-induced APD prolongation (3 out of 3 cells, in 2 explants: Fig. 1B5.6). The second paradigm was applied to another group of 10 DRG neurons (in 4 explants) by initial pretreatment with 10 nm diprenorphine. None of these cells showed any APD alteration when a low (10 nm) or high concentration (1 pm) of DADLE was coperfused with the antagonist. Initial pretreatment of another group of DRG neurons with 10 nm naloxone (n = 10, in 4 explants) also blocked completely the APD prolongation usually elicited by 10 nm DADLE or the APD shortening by 1 /~M DADLE. These results provide strong evidence that APD prolongation elicited by 6-, p- and ~c-opioid agonists in DRG neurons are mediated by specific opioid receptors. Effects of DADLE and U-50,488H on the APD of DRG neurons in the presence of multiple K channel blockers DADLE. Prolongation of the APD after DADLE treatment can be due to increased Ca z+ entry following either an increase in voltage-dependent Ca 2+ conductance or decrease in voltage- or Ca 2+dependent K conductance. To determine which conductance is responsible for the DADLE-induced APD prolongation of DRG neurons, tests were made in the presence of multiple K channel blockers: Ba z+, TEA, Cs, no extracellular Ca 2 (see Materials and Methods). Under these conditions, the APD of DRG neurons was prolonged to values ranging from 9 to 64 ms ( ms, n = 11), in contrast to a mean value of ms (n = 170) in control Ca, Ba/BSS. When exposed to 10 nm DADLE in the presence of this set of K channel blockers, the usual APD prolongation no longer

8 234 occurred in most of the DRG neurons tested (8 out of 9 cells; Table IV). The marked attenuation of DADLE-induced APD prolongation in neurons exposed to multiple K + channel blockers cannot be accounted for simply by the much longer durations of the APs prior to the opioid test. FSK treatment often resulted in even longer AP durations (see below; e.g. Fig. 3B 0, yet DADLE still elicited further prolongation of the APD (Fig. 3B2,3). These results suggest that DADLE-induced prolongation of the APD of DRG neurons may be mediated by opioid receptor subtypes that depress a voltage- or Ca2+-dependent K + conductance. When tested with 1,uM DADLE in the presence of the multiple K channel blockers, APD prolongation occurred in only 1 out of 11 DRG neurons (Table IV), as observed in the tests with 10 nm DADLE. However, 82% of the cells (n = 11) showed APD shortening, in contrast to the complete absence of shortening effects with 10 nm DADLE during K + channel blockade (Table IV). The marked decrease in the proportion of DRG neurons showing APD prolongation by 1/~M DADLE in the presence of multiple K channel blockers (9% vs 34% in control medium) provides further evidence that DADLE-induced APD prolongation may be mediated primarily by opioid receptor subtypes that depress a voltage-sensitive K conductance. In addition, the DADLE-induced APD shortening which is unmasked during tests with 1 ktm DADLE in the presence of multiple K channel blockers (82% of the cells, vs 49% in control medium) may be mediated by another subtype of opioid receptors that decreases a voltage-sensitive Ca 2+ conductance. The latter are presumably inhibitory ~c-receptors, as have been demonstrated in dissociated DRG neurons exposed to dynorphin in the presence of K channel blockers 68. U-50,488H. In contrast to the marked attenuation of DADLE-induced APD prolongation in DRG neurons tested in the presence of multiple K + channel blockers, the proportion of cells showing U-50,488H-induced APD prolongation (78%: Table IV) was not significantly decreased after similar K channel blockade (70%; n = 27). On the other hand, the proportion of cells showing APD shortening in 1 nm U-50,488H was increased from 8% to 26% (Table IV) in the presence of multiple K channel blockers. These results with low concentrations of U-50,488H suggest that APD prolongation elicited by this ~c-agonist is mediated by an excitatory opioid receptor subtype that increases a voltage-sensitive Ca 2+ conductance, in contrast to the inhibitory 1c-receptors that mediate a decrease in voltagesensitive Ca 2 conductance (as noted above; see ref. 68). Altered responsiveness of DRG neurons to DADLE after treatment with PTX PTX is known to prevent the negative coupling of opioid and other inhibitory receptors to adenylate cyclase and other effectors by APD-ribosylation of TABLE IV ln presence of multiple K + channel blockers DADLE butnot U-50,488H fails to prolong APD of DRG neurons Opioid effect Proportions of DRG neurons tested (%) onapd DA DLE(IO nm) b DADLE (1 fim) U-50, 488H (1 nm) Ca, Ba/BSS Multiple K + Ca, Ba/BSS Multiple K + Ca, Ba/BSS Multiple K + (n = 54) channel (n = 74) channel (n = 37) channel blockers a (n = 9) blockers a (n = 11) blockers a (n = 27) Shortened Prolonged Not altered Bathing medium included 5 mm TEA, 5 mm Ba 2+, and no Ca 2+, intracellular recording microelectrode was filled with 2 M CsCI. Significant difference (P < 0.001) was detected between groups tested with 10 nm and 1/~M DADLE, but not with U-50,488H, in the presence of multiple K channel blockers. b Significant difference was detected between groups in Ca,Ba/BSS and multiple K channel blockers when tested with 10 nm DADLE (P < 0.001).

9 235 specific nucleotide-binding proteins, e.g. Gi and Go 25"29'39. TO determine whether pretreatment with PTX would attenuate the inhibitory effect of DADLE on the APD of DRG neurons, DRG-cord explants were exposed to 1 gg/ml PTX for >4-5 days, a dosage which blocks opioid-depression of DRG-evoked dorsal-horn network responses in these cultures t3. PTX treatment resulted in a marked decrease in the proportion of DRG neurons showing DADLE-induced shortening of the APD. Only 16% of PTX-treated DRG neurons showed APD shortening when tested with 1/~M DADLE (5 out of 32 cells in 18 explants; Table V). Moreover, an unusually large proportion of PTX-treated neurons (53%) showed a marked DADLE-induced APD prolongation. These results are significantly different from the opioid responsiveness of naive DRG neurons, where 49% showed DADLE-inhibitory effects and 34% showed DADLE-excitatory effects (Table V). It is evident that PTX treatment of DRG neurons not only attenuates opioid-induced APD shortening by uncoupling opioid receptors from inhibitory GTP-binding proteins (e.g. Gi and Go: see Discussion), but it also enhances opioid-induced APD prolongation. This opioid excitatory effect is apparently Overridden by opioid inhibitory effects when control DRG neurons are tested with higher concentrations of DADLE (ca. 1/~M). of sustained depolarizing currents (100 ms) to the neuronal somas. Of the FSK-treated DRG neurons (n = 32) 69% showed repetitive firing: 2-9 APs per 100 ms pulse, whereas in control media (5 mm Ca2+/BSS), only 9% (n = 43) showed repetitive firing, at lower frequency: 2-3 APs per 100 ms (see further details in ref. 15). When the DRG neurons were treated with FSK (50 gm) in BSS containing 5 mm Ba 2 as well as 5 mm Ca 2+, a much larger increase in the APD occurred in most of the cells, ranging from 100% to 1500% of control values (as observed in chick 22 and mouse 27 DRG neurons in dissociated cell culture). APD prolongation started at 3-5 min after introduction of FSK and progressively increased to a steady state ranging from 6 to 122 ms ( ms; n = 19) by 15 min (Fig. 3Ba). Furthermore, after treatment with FSK in Ca 2+, Ba2+/BSS, some of the cells were so excitable that even a single, brief (2 ms) depolarizing pulse could trigger afterdischarges consisting of 1-3 long-latency action potentials following the initial evoked AP (Fig. 3B; see also ref. 15). In addition to the direct excitatory effects of FSK on DRG neurons, marked alterations occurred in the opioid responsiveness of these FSK-treated neurons. After exposure to FSK (50/~M) for more than 15 min, a marked decrease was found in the Altered responsiveness of DRG neurons to DADLE after treatment with forskolin Forskolin (FSK) activates adenylate cyclase (AC) and markedly increases the intracellular level of cyclic AMP (camp) 51. To determine if the AC/ camp system is involved in opioid-induced APD prolongation and/or shortening of DRG neurons, the cells were exposed to 50/~M FSK, a concentration that markedly attenuates opioid-depressant effects on DRG-evoked dorsal-horn network responses in DRG-cord explants TM. After a 30 min treatment with 50 ~M FSK in 5 mm Ca2+/BSS, no significant alteration of the APD was observed in most of the DRG neurons tested; 5 out of 30 cells showed APD prolongation ranging from % of control values. Nevertheless, many of the FSK-treated DRG neurons showed augmented excitability as evidenced by a significant increase in repetitive firing of APs during application TABLE V Attenuation of opioid-induced APD shortening and enhancement of APD prolongation in DRG neurons pretreated with FSK or PTX PTX: explants were pretreated with 1 #g/ml PTX for >4 days. FSK: cultures were perfused with 50/~M FSK for about 30 min. APDs became prolonged after 3-5 min and reached steady values ranging from 6 to 122 ms ( ms; n = 23) prior to testing with DADLE. Cells in Ca,Ba/BSS and PTX groups were tested with 1 #M DADLE; FSK-treated neurons were tested with 10 gm DADLE (see text). Note: significant difference was detected between the FIX and control groups as well as between FSK and control groups (P < 0.01). DA DLE effect on A PD Proportions (%) of DRG neurons tested with DADLE (1-10 #M) Ca, Ba/BSS P TX FSK (n = 74) (n = 32) (n = 23) Shortened Prolonged Not altered

10 236 A1 " 2 msec B1. FSK C1 " FSK :.L Ca 100 rnsec ----f Ca, Ba (BSS) FSK + lum DADLE C2 ~' FSK + lum DADLE / l A3 - I :~ DADLE lum 13 3" FSK + 10 um DADLE C3 " FSK + DADLE + NAL,P,=,,,. I Fig. 3. FSK treatment reverses opioid responsiveness of another DRG neuron and naloxone blocks DADLE-induced APD prolongation in presence of FSK. A: APD is prolonged after addition of 5 mm Ba 2 to BSS (cf. A 2 vs A1) and is shortened after 1 min exposure to!/~m DADLE (A3). Bl: after withdrawal of opioid and treatment with 50/~M FSK for 20 min, APD is markedly prolonged (note slower sweep rate). Note also AP after-discharge following one of the initial evoked APs (3 superimposed sweeps; see text). B2: addition of 1/~M DADLE to FSK-treated neuron now prolongs APD (6 min test period), in contrast to opioid-induced APD shortening prior to FSK (record A3). The duration of the initial AP in the 3rd sweep is about 60 ms vs 47 ms prior to DADLE (cf. B,) and the duration of the long-latency secondary AP is similarly increased (cf. B 0. B3: after increasing DADLE concentration to l0 #M, the APDs of the initial as well as the after-discharge responses are still further prolonged (16 min test period; duration of initial AP in 3rd sweep is now about 74 ms). C~: FSK (50/~M) prolongs APD in another DRG neuron, as in record B t (10 min exposure). C2: addition of 1/tM DADLE to FSK-treated neuron elicits marked prolongation of APD (5 min test period). C3: coperfusion of 3/~M naloxone with DADLE reverses the DADLE-induced prolongation of the APD (by 2 min). proportion of DRG neurons in which DADLE shortened the APD. Only 26% of the FSK-pre- treated DRG neurons (6 out of 23 cells) showed APD shortening in 10 pm DADLE (Table V), in contrast to 49% of control cells (n = 74) which showed DADLE-induced APD shortening. On the other hand, the proportion of FSK-treated DRG neurons that showed APD prolongation in 10 ~M DADLE increased to 70% (16 out of 23 cells, Table V), in contrast to only 34% of control cells (n = 74). A 10-fold higher concentration of DADLE was generally used for tests after FSK treatment (i.e. 10 /~M vs 1 #M) in order to ensure detection of residual DADLE-induced inhibitory effects and to challenge more effectively the expression of DADLE-induced excitatory effects. Tests with naloxone were made in order to confirm that the enhancement of APD prolongation by DADLE in FSK-treated DRG neurons was mediated by opioid-receptors rather than by a progressive increase in direct excitatory effects of FSK. Naloxone (3 ~tm) reversed the DADLE- induced APD prolongation in 4 out of 5 tests on FSK-pretreated DRG neurons (Fig. 3C3).

11 237 Since the alteration of opioid responsiveness of DRG neurons developed much more rapidly during treatment with FSK as compared to PTX, it was possible to study the reversal of opioid-induced APD shortening effects in the same DRG neurons before and after FSK treatment. Tests were made on 19 DRG neurons in 14 DRG-cord explants. Twelve of the DRG neurons showed APD shortening when tested in i/~m DADLE (mean decrease in APD: %). In contrast, after rinsing in Ca,Ba/BSS and treating with 50/~M FSK for 15 min, 9 of these cells showed a reversed opioid responsiveness, i.e. the APD was prolonged (by 48 to 148 ms; mean increase in APD %) when retested in 10/~M DADLE (Fig. 3B2. 3 vs B1). One of the neurons showed attenuation of DADLE-induced APD shortening and two showed no significant change in opioid responsiveness. Five DRG neurons which had shown DADLE-induced APD prolongation prior to FSK treatment ( % of controls) showed a marked (2-3 fold) enhancement in the magnitude of DADLE-induced APD prolongation after FSK. The reversal of DADLE-induced APD shortening to APD prolongation in 9 out of 12 FSK-treated DRG neurons and the augmentation of DADLE excitatory effects on the APD of 5 other cells demonstrate that elevation of AC/cAMP levels can rapidly enhance excitatory opioid responsiveness in DRG neurons. DISCUSSION The present study demonstrates that d-, ~- and ~:-opioid agonists produce naloxone-reversible, concentration-dependent dual modulation of the Ca 2+ dependent component of the AP in many DRG neurons: low opioid concentrations prolong the APD, higher concentrations shorten the APD. These results confirm and extend previous observations by Higashi et al. 3 of dual modulatory effects of morphine on the APD of nodose ganglion neurons in the adult rabbit (see Introduction). In a recent study of dissociated fetal mouse DRG neurons, we found that more cells responded with APD prolongation when exposed to low concentrations of DADLE (10 nm), whereas APD shortening occurred in higher concentrations of DADLE (1 /~M) 8. Furthermore, both the excitatory and inhibitory effects were prevented by coperfusion with the opioid antagonist, diprenorphine. These results in monolayer cultures of isolated DRG neurons, devoid of spinal cord neurons and non-neuronal cells, are in good agreement with the data reported in the present study of DRG neurons in organotypic DRG-cord explants. They also confirm and extend a preliminary report by Werz and Macdonald 64 that low concentrations of Met-enkephalin prolonged the APD of dissociated fetal mouse DRG neurons, whereas higher concentrations shortened the APD. Opioids can shorten the APD of DRG neurons when tested in normal physiological salt solutions, although the effects are less dramatic than observed in the presence of some types of K + channel blockers i.e. Ba 2 or TEA 6"45' In contrast, opioid- induced APD prolongation in sensory neurons has been demonstrated only when the BSS was supplemented with Ba 2 or TEA (in the present study as well as in the report by Higashi et al.3 ). FSKinduced APD prolongation also appears to be dependent on the presence of Ba 2 (refs. 15, 22) or TEA 27. These results suggest that attenuation of camp-dependent voltage-sensitive K conductance by opioids or FSK may be effective in prolonging the APD of DRG neurons only when certain populations of K channels are blocked. Perhaps, in situ, severe nociceptive stimuli 36 or other stressful conditions 6 (see below) may result in K + channelblocking effects that are mimicked by Ba 2 or TEA in vitro. Such effects might be elicited in situ by increased extracellular levels of bradykinin, histamine, corticotropin releasing factor 3'31'63 and/or elevated intracellular ATP levels 4'57, thereby enhancing opioid-induced APD prolongation in sensory neurons. Our in vitro results show interesting correlations with studies by Kayser et al. 36, who used an animal model of persistent pain (arthritic rats), in which 'exceedingly low doses of morphine... elicit a naloxone-reversible paradoxical hyperalgesia', whereas increased doses are 'highly effective in producing analgesia'. Furthermore, single-unit recordings from dorsal-horn neurons in normal rats showed that application of low concentrations of/~or ~c-opioids to the spinal cord produced facilitation of C-fiber-evoked nociceptive responses, whereas higher concentrations resulted in inhibition 18'38.

12 238 These excitatory effects of tow concentrations of opioids on nociceptive transmission in normal, as well as in arthritic, rats are remarkably consonant with our in vitro evidence of APD prolongation in DRG neurons by nm levels of opioids. Mediation of opioid-induced APD prolongation of DRG neurons by excitatory subtypes of opioid receptors Our data demonstrate that (1) opioid-induced APD prolongation of DRG neurons can be elicited by specific 6-,/~- and ~c-receptor agonists at low (nm) concentrations (Tables I-III; Figs. 1-2); (2) APD prolongation elicited by DADLE, DAGO, or U- 50,488H in DRG neurons is prevented in the presence of specific opioid antagonists, naloxone or diprenorphine. These results indicate that opioidinduced prolongation of the APD is mediated by high-affinity opioid receptors. Opioid-induced shortening of the APD of DRG and other neurons has been shown to be mediated by inhibitory subtypes of opioid receptors (e.g. refs. 6, 45, 46, 66-68). We postulate that opioids prolong the APD of DRG neurons by activating excitatory subtypes of opioid receptors. However, our electrophysiologic data do not preclude possible conformational changes in opioid receptors that may result in coupling to inhibitory G proteins (e.g. Gi/Go) when exposed to high opioid concentrations, whereas the same receptor subtypes might switch their linkage to stimulatory G proteins (e.g. Gs) 16 in the presence of low opioid concentrations. Nevertheless, dual opioid modulation of the APD of DRG neurons by distinct subtypes of excitatory and inhibitory receptors is a reasonable hypothesis, especially in view of the well-established dual modulatory effects elicited by excitatory and inhibitory subtypes of monoaminergic, muscarinic and purinergic receptors, e.g. beta- vs alphae-noradrenergic 24' 35,49,50 and A~ vs A 2 adenosine 2 receptors. Ionic conductances mediating opioid-induced APD prolongation Tests carried out in the presence of multiple K + channel blockers indicate that DADLE-induced APD prolongation may be mediated primarily by excitatory opioid receptor subtypes that decrease a voltage-sensitive K conductance (Table IV). In preliminary patch-clamp studies of dissociated fetal mouse DRG neurons in culture, we have obtained more direct evidence that nm concentrations of selective opioid agonists can block specific outward K + currents (S.-F. Fan, K.-F. Shen and S.M. Crain, in preparation). Our data are in good agreement with studies on other types of neurons where excitatory monoaminergic receptor subtypes have been shown to decrease specific voltage-sensitive K + conductances via enhancement of AC/cAMP levels, e.g. serotonergic receptors on Aplysia sensory ganglion cells 5,37'56 and fl-noradrenergic receptors on rat hippocampal neurons 4. In contrast, the APD-shortening effects elicited by 10 nm DADLE that totally disappeared in the presence of multiple K + channel blockers are probably mediated by inhibitory opioid receptor subtypes that increase a voltage-dependent K + conductance. These results are similar to the /~-/6-opioid-induced shortening of the APD of DRG neurons in dissociated cell cultures which is blocked after intracellular injection of Cs + (ref. 68). On the other hand, the APD-shortening effects elicited by higher concentrations of DADLE (1 ~M) that were unmasked in the presence of multiple K + channel blockers appear to be mediated by inhibitory opioid receptor subtypes that decrease a voltage-sensitive Ca 2+ conductance. These results are similar to the APD shortening elicited by the ~-opioid agonist, dynorphin in dissociated DRG neurons which was not attenuated during K + channel blockade 68 and also to the APD shortening by the specific ~c-agonist, U-50,488H (1 rim) in DRG cells of our DRG-cord explants (Table IV) which appeared to be unmasked in the presence of multiple K + channel blockers. The fact that U-50,488H-induced APD prolongation still occurred in the presence of multiple K + channel blockers suggests that this ~c-agonist may activate excitatory opioid receptor subtypes that increase a voltage-sensitive Ca z+ conductance (as occurs, for example, in hippocampal granule neurons exposed to fl-adrenergic agonists26). Thus opioid excitatory modulation of the APD appears to be mediated by receptor subtypes that produce the opposite effects on voltage-sensitive K + and Ca z+ channels as those demonstrated by Werz and Macdonald to occur during opioid-induced APD shortening.

13 239 Pertussis toxin blocks opioid inhibitory effects and enhances opioid excitatory effects in DRG neurons PTX treatment of DRG-cord explants resulted in a marked decrease in the proportion of DRG neurons in which DADLE shortened the APD and also a prominent increase in the fraction of DRG neurons showing DADLE-induced APD prolongation. These results are in good agreement with our previous evidence that PTX-treatment of DRGcord explants not only blocked the depressant effect of opioid agonists on DRG-evoked dorsal-horn synaptic-network responses but also resulted in opioid enhancement of the amplitude of dorsal horn responses in some of the cultures 13. Data from biochemical assays of DRG-cord explants after similar treatment with PTX are consonant with our electrophysiological findings. In PTX-treated explants, the inhibitory (Gi-mediated) effect of opioids on FSK-stimulated AC was attenuated, whereas the stimulatory effect of opioids on basal AC activity was enhanced 41. These electrophysiological and biochemical data suggest that PTX-sensitive G proteins may be involved in the opioid inhibitory effects on DRG neurons. However, there is no compelling evidence that opioid inhibition of AC/cAMP levels is directly responsible for the opening of K or closure of Ca e+ channels that result in shortening of the APD of DRG neurons 2,19'43'47. Opioid inhibitory receptors on these neurons may be linked via other PTXsensitive G proteins to second messengers other than camp 23'25'29'34 or directly to ion channels as occurs in locus coeruleus neurons 43. On the other hand, the increase in the proportion of DRG neurons which showed opioid-induced APD prolongation after PTX treatment may be due to the unmasking of pre-existing opioid excitatory effects following PTX-blockade of G protein-mediated opioid-induced APD shortening in the same neuron. In addition, PTX blockade of Gi-mediated opioid inhibition of AC may result in more effective Gs-mediated opioid stimulation of a common pool of AC by excitatory opioid receptor subtypes (see below). The elevated levels of camp may in turn close phosphorylation-dependent, voltage-sensitive K channels (or open voltage-sensitive Ca 2 channels), as suggested by our whole-cell recordings from DRG neurons in dissociated cell cultures where intracellular dialysis with an inhibitor of campdependent protein kinase selectively blocked opioidinduced prolongation of the APD 8. Forskolin enhances opioid excitatory effects in DRG neurons DADLE-induced excitatory effects were greatly enhanced after FSK treatment. In DRG neurons where 1 /~M DADLE prolonged the APD when tested in Ca, Ba/BSS, much larger opioid-induced APD prolongation occurred after FSK treatment. These DRG neurons may have been similar to those which responded with APD prolongation to both high as well as low (10 nm) concentrations of DADLE (Table II: group D). In another group, of DRG neurons where 1/~M DADLE shortened the APD in Ca, Ba/BSS, a reversal to opioid-induced APD prolongation occurred after FSK treatment. These neurons may have similar properties to those which showed APD prolongation at low DADLE concentration and APD shortening at high concentration (Table II: group A). Such alterations of opioid responsiveness may be due to elevation of AC activity in FSK-treated neurons, thereby neutralizing Gi-mediated opioid inhibition of AC and facilitating or unmasking Gs-mediated stimulation of AC via excitatory opioid receptors s (see above). These results provide a particularly dramatic demonstration that the opioid responsiveness of some DRG neurons can be rapidly switched from an inhibitory to excitatory mode by an increase in AC/cAMP levels. A third group of DRG neurons continued to show DADLE-induced APD shortening even after FSK treatment. The lack of effect of FSK on the opioid responsiveness of these groups of cells may have been due to a relatively high proportion of inhibitory opioid receptors that were coupled to camp-independent conductances (Table II: groups B and C). Physiologic significance of excitatory and inhibitory modulation of the APD in DRG neurons Opioids have been shown to inhibit release of substance P from DRG neurons in dissociated cell cultures at concentrations similar to those which shorten the APD of the perikarya of these cells TM (see also ref. 33). These results suggest that opioid shortening of the APD of DRG perikarya may

14 240 provide a useful model of opioid inhibition of calcium influx and transmitter release at presynaptic DRG terminals ~j6.zs4~''65'66 (see also ref. 37). In contrast, if excitatory opioid receptors are present on some types of presynaptic DRG terminals, they may prolong the APD in these terminals (as occurs in DRG perikarya in the present study) and increase transmitter release 1~'~6 (analogous to the effects mediated by excitatory serotonergic receptors on the perikarya and terminals of Aplysia sensory ganglion neurons 5.37). Recent studies provide significant support for our hypothesis that opioids may prolong the APD in some DRG terminals, thereby enhancing transmitter release. Mauborgne et al. 42 and Pohl et al. 48 have demonstrated that exposure of rat dorsal spinal cord slices to /~-opioids results in naloxone-reversible enhancement of the capsaicin-evoked release of substance P from primary afferent (presumably nociceptive) fibers. Sweeney et al. 5~'59 have shown that morphine enhances Ca2+-dependent release of endogenous adenosine from rat dorsal spinal cord synaptosomes in vitro and from capsaicin-sensitive primary afferent terminals in the spinal cord in vivo, both of which are blocked by the opioid antagonist, naltrexone. Furthermore, a preliminary report by Gintzler and associates 69 indicates that opioid modulation of electrically evoked enkephalin release from enteric ganglia is bimodal: low (nm) concentrations of specific kt-, 6- and 7c-opioid agonists enhance enkephalin release, whereas higher concentrations decrease release. In addition, after FSKtreatment of these ganglia the usual inhibitory modulation of enkephalin release by high opioid concentrations is reversed to an enhancement of release (A.R. Gintzler, personal communication). These data are in excellent agreement with the enhanced opioid excitatory modulation of the APD that we-have observed in FSK-treated DRG neurons. The results of the present study together with the correlative evidence noted above have led us to postulate that critically distributed excitatory and inhibitory opioid receptors on DRG neurons may provide integrative presynaptic opioid modulation of nociceptive and perhaps other primary afferent networks in the CNS. They may also mediate some of the paradoxical hyperalgesic and aversive effects of opioids 36'6 '61 in the central and peripheral nervous system. Dual opioid modulation of the APD of DRG neurons may also provide insights into tolerance and plasticity in opioid networks. In a recent study of similar DRG-cord explants after chronic exposure to 1,uM DADLE, we found that most DRG neurons became tolerant to the usual APD-shortening effects of high concentrations (10 /~M) of DADLE. In addition, a much larger proportion of the treated cells showed APD prolongation in response to an acute increase in the DADLE level TM. These results are remarkably similar to the alterations observed in the present study after PTX or FSK treatment. In all 3 cases, a net increase in the efficacy of excitatory opioid receptor-mediated functions appears to occur in the treated DRG neurons. Our in vitro analyses may therefore provide clues to compensatory processes (possibly mediated by enhanced AC/cAMP level 5"1 A2"14"16"41"52"53) that could attenuate or block opioid inhibitory effects on primary afferent synaptic networks in the spinal cord, thereby elucidating mechanisms underlying tolerance to opioid analgesia in situ. ACKNOWLEDGEMENTS This work was supported by Research Grants DA and DA to S.M.C. The cultures were prepared by Edith R. Peterson, Elena Pousada and Peter Vanamee in facilities kindly provided by Dr. M.B. Bornstein. We thank Drs. A. Chalazonitis and G.-G. Chen for advice and discussion during the course of this study. REFERENCES 1 Aghajanian, G.K. and Wang, Y.-Y., Pertussis toxin blocks the outward currents evoked by opiate and alpha2-agonists in locus coeruleus neurons, Brain Research, 371 (1986) Aghajanian, G.K. and Wang, Y.-Y., Common alpha,- and opiate effector mechanisms in the locus coeruleus: intracellular studies in brain slices, Neuropharmacology, 26 (1987) Aldenhof, J.B., Gruol, D.L., Rivier, J., Vale, W. and Siggins, G.R., Corticotropin releasing factor decreases postburst hyperpolarizations and excites hippocampal neurons, Science, 221 (1983)

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