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 10461 (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. 0.1-1/~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 general ly produce inhibi tory modula t ion
of nerve cells e i ther by membrane hyperpolar iza t ion
that decreases the firing rate of action potent ials
(e.g. in locus coeruleus neurons a6) or by shortening
the action potent ia l dura t ion (APD) (e.g. in dorsal root ganglion ( D R G ) neurons of the mouse 6'65-68
and chick45). These and re la ted opioid inhibitory
effects are b locked by pertussis toxin (PTX) which
uncouples various types of opioid and o ther modu-
latory receptors from regula tory G prote ins (e.g. Gi and GO) 1'2'13'29'34'43. Opioid inhibi tory modula t ion
of the A P D of D R G per ika rya is media ted by: (1)/~-
and 6-0pioid receptors that increase voltage- and/or
ca lc ium-dependent K÷-conductances , and (2) r -
opioid (dynorphin) receptors that decrease a volt- age-dependent Ca2+-conductance 66-68 (see also ref.
9). It has been pos tu la ted that similar opioid- induced
opening of K ÷ channels and/or closing of Ca 2÷
channels in presynapt ic D R G terminals may result in
shortening of the A P D , reduct ion in Ca 2÷ influx and decrease in neuro t ransmi t te r release 45,46,66-68.
In addi t ion to these opioid inhibi tory effects on
the A P D of D R G neurons , we have found that
specific 6-,/~- and r -op io id 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.
the APD especially when applied at low concentra- tions (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 (10-100 nM) the APD was shortened. These effects of morphine were prevented in the presence of naloxone 3°. The pres- ent 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 prolonga- tion 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 inter- neurons 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-co rd 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 con-
taining 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 tetraethylammo- nium chloride (TEA) prior to introduction of opioids (as in studies by Werz & Macdonald 66-68 and Higashi et al.3°). To prevent Ba 2+ and Ca 2+ precipitation,
the usual bicarbonate/phosphate buffers were re- placed 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 monitor- ing. 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 60-100 MI2) and were con- nected via a chloridized silver electrode to a neu- tralized input capacity preamplifier (WPI-M707A). A bridge circuit allowed simultaneous current pas- sage and voltage recording with a single microelec- trode. After impalement of a D R G 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 con- centrations that have been shown to be effective on DRG neurons 45'6s (see also refs. 46, 47): 5 mM
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 experi- ments 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 con- sidered 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 mea- sured 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 = penicilla- mine; 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 pro- longation or shortening, or no effect, we used a )~2 test of contingency.
RESULTS
Dual effects o f D A D L E on the A P D 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 D R G - cord explants. The magnitude of APD shortening produced by 1/aM DADLE in these DRG neurons ranged from -17 to -81% (mean + S.E.M.: -40 + 3%; 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% (+70 + 9%; 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. 50-100 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
230
A ~ _ A2__ A3_ BalSSS DAOLE DADLE
lOnM luM
D I P R E N O R P H I N E (1OHM) P R E T R E A T M E N T
A 4 - A 5 - A 6 - DAOLE DADLE
lOnM luM
@ I.,. B 2 . . B 3 ~ TEAIBSS DADLE DADLE
1OhM luM
-_/ - / -
NALOXONE (1OHM) P R E T R E A T M E N T
B4_ BS~ B% DADLE OADLE
1OhM 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 nalox- one. 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 prolon- gation 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 D A D L E 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.
D A D L E effect Proportions (%) o f DRG neurons tested on A PD . . . . .
* In x 2 tests, P < 0.001 vs 1 #M or 10ktM DADLE groups.
not causally re la ted to the o b s e r v e d op io id - induced
A P D p ro longa t ion o r shor ten ing . T h e R m of the
D R G neurons r anged f r o m 22 to 126 Mg2 ( m e a n 70
+ 8.5 Mg2; n = 18) and of ten d e c r e a s e d dur ing
exposure to 10 n M D A D L E (by 11 to 6 2 % ; m e a n 35
+ 4 . 5 % , in 13 out of 18 cells). H o w e v e r , the
dec rease in Rr, showed no co r re l a t ion with opio id-
induced A P D al tera t ions .
O p i o i d - i n d u c e d A P D shor t en ing was readi ly re-
ve rsed within a few minu tes by severa l successive
rinses of the ba th wi th BSS. In cont ras t , opio id-
induced A P D p ro longa t i on of ten r eve r sed re la t ive ly
slowly dur ing per iods o f 5 -15 min af ter op io id
wi thdrawal , as o b s e r v e d in s imilar tests on chron ic
D A D L E - t r e a t e d D R G neu rons ~6. A second acute
op io id exposure was of ten e f fec t ive in p ro long ing the
A P D again w h e n appl ied af ter a 10-15 min BSS
rinse (21 ou t of 29 cells).
Preferent ia l A P D p r o l o n g a t i o n o f D R G n e u r o n s
e x p o s e d to l ow concen t ra t ions o f D A D L E
A much larger p r o p o r t i o n of D R G neurons
showed A P D p ro longa t ion in 10 n M D A D L E (76%;
e.g. Fig. 1A2, B2), and only 9 % s h o w e d A P D
shor ten ing (n = 54; Table I). T h e m a g n i t u d e of the
A P D p ro longa t ion el ic i ted by 10 n M D A D L E
ranged f rom + 1 3 % to + 4 0 0 % ( + 4 7 + 10%) . Thus ,
10 n M D A D L E - i n d u c e d A P D p r o l o n g a t i o n in s o m e
D R G neurons was c o m p a r a b l e to , or l a rger than, the
pe rcen t age increase e l ic i ted by 1 ~ M D A D L E ,
a l though the m e a n increase in A P D was smal ler , i .e.
+ 4 7 % vs + 7 0 % . A n addi t iona l ser ies of D R G
A
. , , .J B S S
B C D
. - . . . . . / _ .-._i BSS BSS BSS
231
. . . . .
D P D P E lOnM U - 5 0 4 8 8 H lOnM DAGO 10nM DADLE 100nM
D P D P E lOOnM U - 5 0 4 8 8 H luM D A G O 1 uM D A D L E 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 D R G - c o r d
explants were tested with 10/~M D A D L E . Similar to
the 1 #M D A D L E series (see above), 55% showed A P D shortening and 24% showed A P D prolonga-
tion (n = 42; Table I). These results demonstrate that DADLE- induced prolongation of the A P D of
To confirm these dose-response effects of D A D L E on the A P D of D R G neurons, 3 different concentrations of D A D L E (10 nM, 100 nM and 1
~M) were tested sequentially on the same cell. A total of 20 D R G neurons were studied (Table II). In
group A, a dual response to different concentrations was obtained (10 cells), where a low D A D L E concentration (10 nM) elicited A P D prolongation and higher concentrations (100 nM and 1 #M)
elicited A P D shortening (e.g. Fig. 1A2,3; Be,3). The magnitude of A P D shortening was concentration- dependent: -26 + 4% and -40 + 4% of the control value in 100 nM and 1 #M D A D L E , respectively. In
groups B and C only A P D shortening occurred
depending on the D A D L E concentration (5 cells).
In group D, only A P D prolongation occurred when
tested at all three D A D L E concentrations (5 cells).
In 3 of these cases, the A P D was progressively
increased at higher D A D L E concentrations (100
nM-1 ~M) (see also similar dose-dependent effects
TABLE II
Dose-dependence o f opioid-induced alterations o f 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. o f cells Effect o f D A D L E 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
232
of DPDPE: Fig. 2A).
High and low concentrations of DADLE were also effective in eliciting APD shortening and pro- longation in DRG neurons when the 5 m M B a 2+ in the BSS was replaced by 5 mM TEA. Low concen- trations of DADLE (10 nM) elicited APD prolon- gation 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 pro- longation has been observed in tests made in the absence of Ba 2+ or TEA.
Effect o f specific opioid agonists on A P D o f D R G
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 acti- vated. 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). More- over, the mean value of the APD prolongation in 10 nM DPDPE was +59 -+ 13% (n = 15), comparable
to that observed in 10 nM DADLE. The specific/~-agonist, D A G O 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 D A G O was +53 + 9% (;7 = 28). Furthermore, 22 of the cells showing DAGO-induced APD pro- longation were also tested with a higher concentra- tion of D A G O (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 D A G O can elicit dual modulatory effects as observed with DADLE, i.e. prolongation of the APD at low concentration (10 riM) and reversal to APD short- ening 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 pro- longation (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 +99 + 14% (n = 29), substantially larger than elicited by
DADLE, D A G O or DPDPE. These tests with low (riM) concentrations of
specific opioid agonists raise the possibility that APD prolongation of DRG neurons may be medi- ated by high-affinity, excitatory subtypes of d-, #- and ~c-opioid receptors, in contrast to the lower- affinity 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, D A D L E 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 sus- tained 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)
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 respon- siveness when tested with specific opioids in differ- ent 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 mem- brane.
Effect of specific antagonists on opioid-induced A P D 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 recov- ery 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 pro- longation when exposed to a low concentration of D A D LE (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 10-15 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 coper- fusion 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 pretreat- ment 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 D A D L E and U-50,488H on the A P D 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 condi- tions, the APD of DRG neurons was prolonged to values ranging from 9 to 64 ms (32 + 6 ms, n = 11), in contrast to a mean value of 11 + 1 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
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 prolon-
gation 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 neu-
rons 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 n o t 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 voltage-
sensitive Ca 2÷ conductance (as noted above; see ref.
68).
A l t e r e d r e spons i venes s o f D R G n e u r o n s to D A D L E
af ter t r e a t m e n t wi th P T X
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 o f multiple K + channel blockers D A D L E butnot U-50,488H fails to prolong A P D o f D R G neurons
Opioid effect Proportions o f DRG neurons tested (%) o n A P D
DA DLE(IO nM) b D A D L E (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)
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).
specific nucleotide-binding proteins, e.g. Gi and Go 25"29'39. TO determine whether pretreatment with
PTX would attenuate the inhibitory effect of D A D L E on the APD of D R G neurons, DRG-co rd 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 D R G neurons showed APD short- ening when tested with 1/~M DADLE (5 out of 32 cells in 18 explants; Table V). Moreover, an unusu- ally large proportion of PTX-treated neurons (53%) showed a marked DADLE-induced APD prolonga- tion. 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 Discus- sion), but it also enhances opioid-induced APD prolongation. This opioid excitatory effect is appar- ently Overridden by opioid inhibitory effects when control D R G neurons are tested with higher con- centrations of D A D L E (ca. 1/~M).
Al tered responsiveness o f D R G neurons to D A D L E
after treatment with forsko l in
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 concentra- tion that markedly attenuates opioid-depressant ef- fects on DRG-evoked dorsal-horn network re- sponses in D R G - c o r d 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 D R G neurons tested; 5 out of 30 cells showed APD prolongation ranging from 114-183% of control values. Nevertheless, many of the FSK-treated D R G neurons showed augmented excitability as evidenced by a significant increase in repetitive firing of APs during application
235
of sustained depolarizing currents (100 ms) to the
neuronal somas. Of the FSK-treated D R G 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 D R G 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 intro- duction of FSK and progressively increased to a steady state ranging from 6 to 122 ms (22 + 6 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 con- sisting 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
TABLE V
Attenuation of opioid-induced APD shortening and enhance- ment 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 (22 + 6 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 D A D L E (1-10 #M)
,P ,= , , , . I Fig. 3. FSK treatment reverses opioid responsiveness of another DRG neuron and
C3 " FSK + DADLE + NAL
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).
proport ion of D R G neurons in which D A D L E
shortened the APD. Only 26% of the FSK-pre-
treated D R G neurons (6 out of 23 cells) showed
APD shortening in 10 pM D A D L E (Table V), in
contrast to 49% of control cells (n = 74) which
showed DADLE- induced APD shortening. On the
other hand, the proport ion of FSK-treated D R G
neurons that showed APD prolongation in 10 ~M
D A D L E 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 D A D L E 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 A P D prolongation
by D A D L E in FSK-treated D R G neurons was
mediated by opioid-receptors rather than by a
progressive increase in direct excitatory effects of
FSK. Naloxone (3 ~tM) reversed the D A D L E -
induced A P D prolongation in 4 out of 5 tests on
FSK-pretreated D R G neurons (Fig. 3C3).
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: 38 + 5%). 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 +314 + 122%) when retested in 10/~M D A D L E (Fig. 3B2. 3 vs B1). One of the neurons showed attenuation of DADLE-induced APD short- ening and two showed no significant change in opioid responsiveness. Five DRG neurons which had shown DADLE-induced APD prolongation prior to FSK treatment (133-300% 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 D R G 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, con- centration-dependent dual modulation of the Ca 2+ dependent component of the AP in many D R G neurons: low opioid concentrations prolong the APD, higher concentrations shorten the APD. These results confirm and extend previous observa- tions 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 concentra- tions of D A D L E (10 nM), whereas APD shortening occurred in higher concentrations of DADLE (1
/~M) 8. Furthermore, both the excitatory and inhibi-
tory effects were prevented by coperfusion with the opioid antagonist, diprenorphine. These results in monolayer cultures of isolated D R G neurons, de-
void of spinal cord neurons and non-neuronal cells, are in good agreement with the data reported in the present study of D R G 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 D R G 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 T E A 6"45'65-67. In contrast, opioid-
induced APD prolongation in sensory neurons has been demonstrated only when the BSS was supple- mented with Ba 2÷ or TEA (in the present study as well as in the report by Higashi et al.3°). FSK-
induced 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 D R G neurons only when certain popula- tions of K ÷ channels are blocked. Perhaps, in situ, severe nociceptive stimuli 36 or other stressful con-
ditions 6° (see below) may result in K + channel- blocking effects that are mimicked by Ba 2÷ or TEA
in vitro. Such effects might be elicited in situ by increased extracellular levels of bradykinin, hist- amine, corticotropin releasing fac tor 3'31'63 and/or
elevated intracellular ATP levels 4'57, thereby enhan-
cing opioid-induced APD prolongation in sensory neurons. Our in vitro results show interesting cor- relations 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.
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 opioid- induced prolongation of the APD is mediated by high-affinity opioid receptors. Opioid-induced short- ening 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 recep- tor subtypes might switch their linkage to stimula- tory 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 mod- ulatory effects elicited by excitatory and inhibitory subtypes of monoaminergic, muscarinic and purin- ergic receptors, e.g. beta- vs alphae-noradrenergic 24' 35,49,50 and A~ vs A 2 adenosine 2° receptors.
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 gan- glion cells 5,37'56 and fl-noradrenergic receptors on rat hippocampal neurons 4°. In contrast, the APD-short- ening 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 inhibi- tory opioid receptor subtypes that decrease a volt- age-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 + chan- nel 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 prolonga- tion 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 neu- rons exposed to fl-adrenergic agonists26). Thus opi- oid 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 Mac- donald 66-68 to occur during opioid-induced APD shortening.
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 prolonga- tion. These results are in good agreement with our previous evidence that PTX-treatment of D R G - cord 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 con- sonant with our electrophysiological findings. In PTX-treated explants, the inhibitory (Gi-mediated) effect of opioids on FSK-stimulated AC was atten- uated, 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 in- volved 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 n e u r o n s 2,19'43'47. Opioid inhibitory receptors
on these neurons may be linked via other PTX- sensitive G proteins to second messengers other than c A M P 23'25'29'34 o r 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 opi- oid-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÷ chan- nels), as suggested by our whole-cell recordings from DRG neurons in dissociated cell cultures where
intracellular dialysis with an inhibitor of cAMP-
dependent protein kinase selectively blocked opioid- induced 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 concen- tration (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 demonstra- tion 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 cel l s TM
(see also ref. 33). These results suggest that opioid shortening of the APD of DRG perikarya may
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 mod-
ulation of electrically evoked enkephalin release from enteric ganglia is bimodal: low (nM) concen- trations of specific kt-, 6- and 7c-opioid agonists enhance enkephalin release, whereas higher concen- trations decrease release. In addition, after FSK- treatment 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 neu- rons. 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 ner-
vous 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 D A D L E 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 func- tions appears to occur in the treated DRG neurons. Our in vitro analyses may therefore provide clues to compensatory processes (possibly mediated by en- hanced AC/cAMP l eve l 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-02031 and DA-05203 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) 390-394.
2 Aghajanian, G.K. and Wang, Y.-Y., Common alpha,- and
opiate effector mechanisms in the locus coeruleus: intra- cellular studies in brain slices, Neuropharmacology, 26 (1987) 793-799.
3 Aldenhof, J.B., Gruol, D.L., Rivier, J., Vale, W. and Siggins, G.R., Corticotropin releasing factor decreases postburst hyperpolarizations and excites hippocampal neu- rons, Science, 221 (1983) 875-877.
5 Castellucci, V.E, Nairn, A., Greengard, P., Schwartz, J.H. and Kandel, E.R., Inhibitor of adenosine 3":5"-monophos- phate-dependent protein kinase blocks presynaptic facili- tation in Aplysia, J. Neurosci., 2 (1982) 1673-1681.
6 Chalazonitis, A. and Crain, S.M., Maturation of opioid sensitivity of fetal mouse dorsal-root ganglion neuron perikarya in organotypic cultures: regulation by spinal cord, Neuroscience, 17 (1986) 1181-1198.
7 Chang, H.M., Kream, R.M. and Holz, G.G., Sufentanil, K-agonist U50,488H and D-AlaE-MetS-enkephalinamide in- hibit substance P release from primary sensory neurons, Soc. Neurosci. Abstr., 14 (1988) 712.
8 Chen, G.-G., Chalazonitis, A., Shen, K.-E and Crain, S.M., Inhibitor of cyclic AMP-dependent protein kinase blocks opioid-induced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures, Brain Research, 462 (1988) 372-377.
9 Cherubini, E. and North, R.A.,/~ and r opioids inhibit transmitter release by different mechanisms, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 1860-1863.
10 Collier, H.O.J., Cellular site of opiate dependence, Nature (Lond.), 283 (1980) 625-629.
11 Crain, S.M., Regulation of excitatory opioid responsivity of dorsal root ganglion neurons. In P. Illes and C. Farsang (Eds.), Regulatory Role of Opioid Peptides, VCH, Wein- heim, ER.G., 1988, pp. 186-201.
12 Crain, S.M. and Makman, M.H., Electrophysiologic re- sponses and adenylate cyclase activities of mouse spinal cord-dorsal root ganglion explants rendered tolerant by chronic exposure to morphine or pertussis toxin. In Y.H. Ehrlich, R.H. Lenox, E. Kornecki and W.O. Berry (Eds.), Molecular Mechanisms of Neuronal Responsiveness, Ple- num, New York, 1987, pp. 331-344.
13 Crain, S.M., Crain, B. and Makman, M.H., Pertussis toxin blocks depressant effect of opioid, monoaminergic and muscarinic agonists on dorsal-horn network responses in spinal cord-ganglion cultures, Brain Research, 400 (1987) 185-190.
14 Crain, S.M., Crain, B. and Peterson, E.R., Cyclic AMP or forskolin rapidly attenuates the depressant effect of opioids on sensory-evoked dorsal-horn responses in mouse spinal cord-ganglion explants, Brain Research, 370 (1986) 61-72.
15 Crain, S.M., Shen, K.-F. and Chalazonitis, A., Altered pharmacologic responsivity of opioid-sensitive dorsal root ganglion (DRG) neurons rendered hyperexcitable by ex- posure of DRG-cord explants to forskolin or pertussis toxin. In N. Chalazonitis and M. Gola (Eds.), Inactivation of Hypersensitive Neurons, Liss, New York, 1987, pp. 291-301.
16 Crain, S.M., Shen, K.-E and Chalazonitis, A., Opioids excite rather than inhibit sensory neurons after chronic opioid exposure of spinal cord-ganglion cultures, Brain Research, 455 (1988) 99-109.
17 Dichter, M.A. and Fischbach, G.D., The action potential of chick dorsal root ganglion neurones maintained in cell culture, J. Physiol. (Lond.), 267 (1977) 281-298.
18 Dickenson, A.H., Sullivan, A.F., Knox, R., Zajac, J.M. and Roques, B.P., Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with mu- and delta-opioid receptor agonists in the control of nociception, Brain Research, 413 (1987) 36-44.
19 Dolphin, A.C., Nucleotide binding proteins in signal
241
transduction and disease, Trends Neurosci., 10 (1987) 53-57.
20 Dolphin, A.C. and Prestwich, S.A., Pertussis toxin re- verses adenosine inhibition of neuronal glutamate release, Nature (Lond.), 316 (1985) 148-150.
21 Duggan, A.W. and North, R.A., Electrophysioiogy of opioids, Pharmacol. Rev., 35 (1984) 219-281.
22 Dunlap, K., Forskolin prolongs action potential duration and blocks potassium current in embryonic chick sensory neurons, Pflagers Arch., 403 (1985) 170-174.
23 Dunlap, K., Hoiz, G.G. and Rane, S.G., G proteins as regulators of ion channel function, Trends Neurosci., 10 (1987) 241-244.
24 Gilman, A.G., G proteins and dual control of adenylate cyclase, Cell, 36 (1984) 577-579.
26 Gray, R. and Johnston, D., Noradrenaline and beta- adrenoceptor agonists increase activity of voltage-depen- dent calcium channels in hippocampal neurons, Nature (Lond.), 327 (1987) 620-622.
27 Grega, D.A. and Macdonald, R.L., Activators of adenyl- ate cyclase and cyclic AMP prolong calcium-dependent action potential of mouse sensory neurons in culture by reducing a voltage-dependent potassium conductance, J. Neurosci., 7 (1987) 700-707.
28 Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J. and Smith, C.F.C., Analogues of beta-LPH 61-64 possessing selective agonist activity at mu-opiate receptors, Eur. J. Pharmacol., 70 (1981) 531-540.
29 Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G., The GTP-binding protein, Go regulates neuronal calcium channels, Nature (Lond.), 325 (1987) 445-447.
30 Higashi, H., Shinnick-Gallagher, P. and Gallagher, J.P., Morphine enhances and depresses Ca2+-dependent re- sponses in visceral primary afferent neurons, Brain Re- search, 251 (1982) 186-191.
31 Higashi, H., Ueda, N., Nishi, S., Gallagher, J.P. and Shinnick-Gallagher, P., Chemoreceptors for serotonin, acetylcholine, bradykinin, histamine and gamma-amino- butyric acid on rabbit visceral afferent receptors, Brain Res. Bull., 8 (1982) 23-32.
33 Holz, G.G., Dunlap, K. and Kream, R.M., Characteriza- tion of the electrically evoked release of substance P from dorsal root ganglion neurons: methods and dihydro- pyridine sensitivity, J. Neurosci., 8 (1988) 463-471.
34 Holz, G.G., Rane, S.G. and Dunlap, K., GTP-binding proteins mediate transmitter inhibition of voltage-depen- dent calcium channels, Nature (Lond.), 319 (1986) 670- 672.
35 Illes, P., Mechanisms of receptor-mediated modulation of transmitter release in noradrenergic, cholinergic and sen- sory neurones, Neuroscience, 17 (1986) 909-928.
36 Kayser, V., Besson, J.M. and Guilbaud, G., Paradoxical hyperalgesic effect of exceedingly low doses of systemic morphine in an animal model of persistent pain (Freund's adjuvant-induced arthritis rats), Brain Research, 414 (1987) 155-157.
37 Klein, M., Camardo, J.S. and Kandel, E.R., Serotonin modulates a new potassium current in the sensory neurons that show presynaptic facilitation in Aplysia, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 5713-5717.
242
38 Knox, R.J. and Dickenson, A.H., Effects of selective and non-selective kappa-opioid receptor agonists on cutaneous C-fibre-evoked responses of rat dorsal horn neurones, Brain Research, 415 (1987) 21-29.
39 Kurose. H., Katada, T., Amano, T. and Ui, M., Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholin- ergic, and opiate receptors in neuroblastoma x glioma hybrid cells, J. Biol. Chem., 258 (1983) 4870-4875.
40 Madison, D.V. and Nicoll, R.A., Cyclic adenosine 3", 5"-monophosphate mediates beta-receptor actions of nor- adrenaline in rat hippocampal pyramidal cells, J. Physiol. (Lond.), 372 (1986) 245-259.
41 Makman, M.H., Dvorkin, B. and Crain, S.M., Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin, Brain Research, 445 (1988) 303-313.
42 Mauborgne, A., Lutz, O., Legrand, J.-C., Hamon, M. and Cesselin, E, Opposite effects of delta and mu opioid receptor agonists on the in vitro release of substance P-like material from the rat spinal cord, J. Neurochem., 48 (1987) 529-537.
43 Miyake, M.J., Christie, M.J. and North, R.A., Single potassium channels opened by opioids in rat central neurons. In J. Cros, J.C. Meunier and M. Hamon (Eds.), Advances in the Biosciences, Vol. 75, Int. Narcotics Research Conf., Pergamon, Oxford, U.K., in press.
44 Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yama- mura, H.T., Galligan, J.J. and Burks, R.F., Bis-penicilla- mine enkepbalins possess highly improved specificity to- ward delta opioid receptors, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5871-5874.
45 Mudge, A.W.. Leeman, S.E. and Fischbach, G.D., En- kephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 526-530.
46 North, R.A., Opioid receptor types and membrane ion channels, Trends Neurosci., 9 (1986) 114-117.
47 North, R.A., Williams, J.T., Surprenant, A. and Christie, M.J., Mu and delta receptors belong to a family of receptors that are coupled to potassium channels, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5487-5491.
48 Pohl, M., Mauborgne, A., Bourgoin, S., Benaliel, J.J., Hamon, M. and Cesselin, F., Neonatal capsaicin release treatment abolishes the modulations by opioids of sub- stance P from rat spinal cord slices, Neurosci. Lett., in press.
49 Rodbell, M., The role of hormone receptors and GTP- regulatory proteins in membrane transduction. Nature (Lond.), 284 (1980) 17-22.
50 Sabol, S.L. and Nirenberg, M., Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha- adrenergic receptor. I. Inhibition of adenylate cyclase mediated by alpha-receptors, J. Biol. Chem., 254 (1979) 1913-1920.
51 Seamon, K.B. and Daly, J.W., Forskolin: a unique diterpene activator of cyclic AMP-generating systems, J. Cyclic Nucleotide Res., 7 (1981) 201-224.
52 Sharma, S.K., Klee, W.A. and Nirenberg, M., Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 3092-3096.
53 Sharma, S.K., Werner, A.K. and Nirenberg, M., Opiate- dependent modulation of adenylate cyclase, Proc. Natl.
Acad. Sci. U.S.A., 74 (1977) 3365-3369. 54 Shen, K.-E and Crain, S.M., Specific opioids can prolong
calcium component of action potentials of mouse dorsal root ganglion neurons in culture by decreasing voltage- sensitive potassium conductance, Soc. Neurosci. Abstr., 13 (1987) 768.
55 Shen, K.-F., Chalazonitis, A. and Crain, S.M., Opioids prolong rather than shorten the action potential duration of sensory neurons after treatment of mouse spinal cord ganglion explants with pertussis toxin or forskolin, Soc. Neurosci. Abstr., 12 (1986) 1011.
56 Siegelbaum, S.A., Camardo, J.S. and Kandel, E.R., Serotonin and cAMP close single K + channels in Aplysia sensory neurons, Nature (Lond.), 299 (1982) 413-417.
57 Stanfield, P.R., Nucleotides such as ATP may control the activity of ion channels, Trends Neurosci., 10 (1987) 335-339.
58 Sweeney M.I., White, T.D. and Sawynok, J., Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline, J. Pharmacol. Exp. Ther., 243 (1987) 657-665.
59 Sweeney, M.I., White, T.D. and Sawynok, J., Morphine, capsaicin and K + release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord, J. Pharmacol. Exp. Ther., 248 (1989) 447-454.
60 Van der Kooy, D., Hyperalgesia functions of peripheral opiate receptors. In: Stress Induced Analgesia, Ann. N.Y. Acad. Sci., 467 (1986) 154-168.
61 Van der Kooy, D. and Nagy, J.I., Hyperalgesia mediated by peripheral opiate receptors in the rat, Behav. Brain Res., 17 (1985) 203-211.
62 Von Voigtlander, P.E, Lahti, R.A. and Ludens, J.H., U-50488: a selective and structurally novel, non-/~ 0c) opioid agonist, J. Pharmacol. Exp. Ther., 224 (1982) 7-12.
63 Weinreich, D., Bradykinin inhibits a slow spike afterhy- perpolarization in visceral sensory neurons, Eur. J. Phar- macol., 132 (1986) 61-63.
64 Werz, M.A. and Macdonald, R.L., Morphine and opiate peptides: differential actions on calcium action potentials, Soc. Neurosci. Abstr., 6 (1980) 416.
65 Werz, M.A. and Macdonald, R.L., Opioid peptides de- crease calcium-dependent action potential duration of mouse dorsal root ganglion neurons in cell culture, Brain Research, 239 (1982) 315-321.
66 Werz, M.A. and Macdonald, R.L., Opioid peptides with differential affinity for mu- and delta-receptors decrease sensory neuron calcium-dependent action potentials, J. Pharmacol. Exp. Ther., 227 (1983) 394-402.
67 Werz, M.A. and Macdonald, R.L., Opioid peptides selec- tive for mu- and delta-opiate receptors reduce calcium- dependent action potential duration by increasing potas- sium conductance, Neurosci. Lett., 42 (1983) 173-178.
68 Werz, M.A. and Macdonald, R.L., Dynorphin and neoen- dorphin peptides decrease dorsal root ganglion neuron calcium-dependent action potential duration, J. Pharma- col. Exp. Ther., 234 (1985) 49-56.
69 Xu, H., Smolens, I. and Gintzler, A.R., Opioid can enhance and inhibit the electrically evoked release of methionine-enkephalin from the enteric nervous system, Soc. Neurosci. Abstr., 14 (1988) 402.
70 Zieglgansberger, W., French, E.D., Siggins, G.R. and Bloom, EE. , Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory inter- neurons, Science, 205 (1979) 415-417.
