Dexamethasone-Induced Selective Inhibition of the Central It Opioid Receptor: Functional in Vivo and in Vitro Evidence in Rodents

Br. J. Pharmacol (1994), 113, 1416-1422 '." Macmillan Press Ltd, 1994 Dexamethasone-induced selective inhibition of the central It opioid receptor: functional in vivo and in vitro evidence in rodents

S. Pieretti, A. Di Giannuario, M.R. Domenici, S. Sagratella, *A. Capasso, *L. Sorrentino & 'A. Loizzo

Istituto Superiore di Sanita', V. le Regina Elena 299, 00161, Roma and *Facolta' di Farmacia, Universita' di Salerno, P.a. V. Emanuele 9, 88084, Penta di Fisciano, Fisciano, Salerno, Italy

1 Endogenous corticosteroids and opioids are involved in many functions of the organism, including analgesia, cerebral excitability, stress and others. Therefore, we considered it important to gain inform- ation on the functional interaction between corticosteroids and specific opioid receptor subpopula- tions. 2 We have found that systemic administration (i.p.) of the potent synthetic corticosteroid, dexamethasone, reduced the antinociception induced by the highly selective p agonist, DAMGO or by less selective ft agonists morphine and P-endorphin administered i.c.v.. On the contrary dexamethasone exerted little or no influence on the antinociception induced by a 61 agonist, DPDPE and a 62 agonist deltorphin II. Dexamethasone potentiated the antinociception induced by the K agonist, U50,488. 3 In experiments performed in an in vitro model of cerebral excitability in the rat hippocampal slice, dexamethasone strongly prevented both the increase of the duration of the field potential recorded in CAl, and the appearance and number of additional population spikes induced by A receptor agonists. 4 In both models pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the antagonism by dexamethasone of responses to the 1i opioid agonists. 5 Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the receptor level, while 6 and K receptors are modulated in different ways. Keywords: Dexamethasone, opioid, analgesia, epilepsy, p receptor

Introduction In 1981 the characterisation of the molecular structure of amethasone pretreatment was also able to prevent the Corticotropin Releasing Factor (CRF) was reported (Spiess appearance of CAl epileptiform bursting induced by mor- et al., 1981). The same authors also reported the CRF- phine and DAMGO on rat hippocampal slices. Both these induced release of adrenocorticotrophic hormone ACTH and inhibitory effects were blocked by cycloheximide pretreat- P-endorphin from the anterior pituitary (Vale et al., 1981). ment performed before dexamethasone (Pieretti et al., 1992). P-Endorphin and other natural and synthetic opioids are It is well known that opioids and opioid peptides involved in a series of functions, such as pain, nerve cell administered intracerebroventricularly or intrathecally act at excitability and epilepsy, immunomodulation, stress and A, 6 and K opioid receptors to produce analgesia or seizures others. Analgesia induced by opioids and opioid peptides is a (Millan, 1986; Pasternak, 1987; Hong et al., 1993). In con- well known phenomenon (Millan, 1986) and the role of trast no data are available, to our knowledge, on the effects endorphins in stress has also been studied (Amir et al., 1980). exerted by glucocorticoids on the analgesic and epileptiform Furthermore, in animals, opioids may have pro- and anticon- effects induced by selective opioid agonists. Since our recent vulsant effects (Ramabadran & Bansinath, 1990). Important paper indicated that both effects could be due to an interac- effects are thought to be induced in the same functions by tion of the two drugs on some specific opioid receptors, we ACTH as well, and to a larger extent by corticosteroids. studied whether this could be verified, and, if this was the Many findings indicate that corticosteroids and ACTH play a case, which mechanism of action could be suggested. In this role not only in stress, but also in pain modulation (Lewis et paper two series of experiments were carried out. In the first al., 1980; MacLennan et al., 1982) and in brain excitability series groups of mice were treated with dexamethasone fol- (McEven et al., 1986; Ortolani et al., 1990). A series of lowed by: the highly selective p agonist (DAMGO) or less investigations have indicated that corticosteroids such as dex- selective p agonists (morphine and ,B-endorphin); selective 6, amethasone exert consistent inhibition against the effects agonist (DPDPE); selective 62 agonist (deltorphin II) or selec- induced by morphine on pain sensitivity and hippocampal tive K agonist (U50,488H). The second series of experiments excitability. There is evidence that ACTH and corticosteroids was performed in an in vitro model of hippocampal slices, reduce opioid analgesia (Winter & Flataker, 1951; Chatterjee perfused with various types of opioid drugs, according to a et al., 1982). We reported that dexamethasone pretreatment schedule analogous to the one used for the first series. was able to reduce morphine analgesia both in the hot plate test (Pieretti et al., 1991) and in the tail flick test (Capasso et al., 1992). We also found that dexamethasone pretreatment Methods was able to prevent the appearance of the epileptiform hippocampal electroencephalographic activity induced by central In vivo experiments morphine administration in rabbits. Furthermore, dex- Male CD-l mice (Charles River, Italy) weighing 25-30 g Author for correspondence.A. Loizzo, M.D. Istituto Superiore di were used in the experiments. The animals were housed in Sanita', Via Regina Elena 299, 00161 Roma, Italy. colony cages (5 mice each) with free access to food and DEXAMETHASONE INTERFERENCE IN OPIOID EFFECTS 1417 water. They were maintained in a climate and light controlled as follows (mM): NaCl 122, KH2PO4 0.4, KCI 3, MgSO4 1.2, room (22 ± 1C, 12/12 h dark/light cycle with light on at NaHCO3 25, CaCl2 1.3, and glucose 10 (pH 7.3). The temp- 07 h 00 min at least 7 days before testing. Testing took place erature of the perfusion chamber was maintained at 33 ± 1C. during the light phase. The animals were brought to the test An interval of 60-90 min elapsed between the time the slices room for at least 3 h before testing. Each animal was used were cut and the start of the recording session. Dexametha- only in one experimental session. In all experiments attention sone-21-phosphate, morphine HC1, methadone HCl, morphi- was paid to the ethical guidelines for investigations of experi- ceptin, DAMGO ([D-A a2-N-methyl-Phe4-Gjy5-olJ-enkephalin), mental pain in conscious animals (Zimmerman, 1983). DPDPE ([D-Pen2-D-Pen5]-enkephalin), deltorphin II, U50,488H (trans-(± )-3,4,Dichloro-N-methyl-N-[2-(1I-pyrrolidinyl)-cyclo- Analgesic assay Mice were tested with the hot plate as hexyl]-benzene-acetamide methanesulphate salt) and cyclo- previously reported (Pieretti et al., 1991). Briefly, the device heximide were dissolved in the CSF and bath-applied. (U. Basile, Italy) consisted of a metal plate (25 x 25 cm) Somatic extracellular field potentials (FPs) were recorded heated to a constant temperature, on which a plexiglass through 3 M NaCl-filled glass microelectrodes (1-5 MQ) in cylinder (20 cm diameter x 16 cm height) was placed. The hot the CAl area after electrical stimulation (0.1 Hz, 70 ms, plate was set to 55 ± 0.50C to give a hind paw licking latency 100-200 gA) of the stratum radiatum. Electrical potentials of 15-17 s in controls. The measurement was terminated at were amplified, recorded on tape (Racal 4DS), digitized at the first paw lick or when the latency exceeded the cut-off 10 kHz, and analysed on line, by an ad hoc software package time (60 s). on a PS2 IBM computer on averaged (five consecutive re- Tail flick latency (Capasso et al., 1992) was obtained using cordings) traces. A total of 112 slices were used, one slice for a tail flick unit (U. Basile, Italy) consisting of an infrared each experiment. source (100 W bulb), whose radiant light of adjustable intensity was focused by an aluminized parabolic mirror on a Experimental protocol The drugs were directly added to the photocell. Radiant heat was focused on a blackened spot perfused solution and after 60 min of perfusion their effects 1-2 cm from the tip of the tail, and the latency was recorded were evaluated on: (1) the amplitude of the primary CAl until the mouse flicked its tail away from the heat source. population spike (PPSA); (2) the occurrence of additional Beam intensity was adjusted to give a tail flick latency of epileptiform populations spikes per field potential (AEPS/ 2-3 s in controls. The animals were restrained during the FP); (3) the duration of the field potential (FPD measured trials by means of a plexiglass cylinder (4 cm in diameter and from the first to the last population spike whose amplitude 8 cm long). In order to avoid tissue damage the measurement was higher than 0.2 mV). The following drugs were used: was terminated if the latency exceeded the cut-off time highly selective 1s agonists (DAMGO, morphiceptin), less (10 s). selective 1i agonists (morphine, methadone), a highly selective In all experiments the response latencies were expressed as 62 agonist (deltorphin II), a highly selective 6, agonist percentage of analgesia which were calculated as (post-opioid (DPDPE), a selective K agonist (U50,488H), dexamethasone latency minus pre-drug latency)/(cut off latency minus pre- and the protein synthesis inhibitor, cycloheximide. The fol- drug latency) x 100. lowing treatments were performed: (1) morphine (300 gM), methadone (100 1M), DAMGO (0.5 1M), morphiceptin Drug administration and experimental procedure All drugs (10 tM), DPDPE (0.5 1AM), deltorphin II (0.5 1M), U50,488H used in the experimental sessions were purchased from Sigma (150 1M), dexamethasone (1001M) were perfused for 60 min; Chemical Co (St. Louis, U.S.A.) with the exception of mor- (2) dexamethasone (100 gM) followed 30 min later by per- phine HCO which was purchased from Carlo Erba (Milan, fusion with morphine (3001M), or methadone (100 1M), or Italy). They were dissolved in 0.9% NaCl solution for intra- DAMGO (0.5 gM), or morphiceptin (10 pM), or DPDPE peritoneal (i.p.) administration, or in distilled water for intra- (0.51M), or deltorphin II (0.5gM), or U50,488H (150pM) cerebroventricular (i.c.v.) administration, on the day of test- plus dexamethasone (100lAM) for 60min; (3) cycloheximide ing. These drugs were injected in a volume of 5 ml kg' or (1 1M) was perfused for 30 min; (4) cycloheximide (1 1M) plus 5 fl per mouse, respectively. The i.c.v. injection was per- dexamethasone (100 AM) followed 30 min later by perfusion formed by the method described by Haley & McCormick with morphine (300 1M), or methadone (100 1M) or DAMGO (1957). In the preliminary experiments the time of the peak (0.5 AM) plus dexamethasone (100 pM) for 60 mn. analgesic effect induced by opioid agonists (morphine, DAMGO, P-endorphin, DPDPE, deltophin II, U50,488H) Statistical analysis administered i.c.v. was determined from time-response curves. Thereafter, dose-response curves of various i.c.v. The results obtained in in vivo and in vitro experiments were doses of some opioid agonists alone or in the presence of analysed by using Student's test or by ANOVA. Statistical dexamethasone-21-phosphate (0.1 mg kg-') administered i.p. significance was assigned to a statistical probability of 5% or 30 min before opioid treatment, was determined in the tail less. flick and in the hot plate tests. The ED" and 95% confidence intervals were computed from the dose-response curve by the method of Litchfield & Wilcoxon (1949) with the aid of a Results computer programme. In another series of experiments mice were pretreated with In vivo experiments cycloheximide (15 mg kg-', administered i.p. 2 h before dexamethasone or 2.5 h before opioid agonist) plus dexametha- Figure 1 and Table 1 show that in both the tail flick and hot sone (0.1 mg kg-' administered i.p., 30 min before opioid plate tests, dexamethasone pretreatment was able to induce a agonists), plus the opioid agonist administered i.c.v. at the consistent shift of EDm( towards higher values i.e. to reduce dose inducing 50% analgesia. strongly the analgesic potency of the drugs which have selective or prevalent 1 effects. However, less evident effects were In vitro experiments induced by dexamethasone on the antinociceptive potency of DPDPE, which has prevailing 61 effects. On the contrary, Transverse slices of hippocampus (450,um thick) were pre- dexamethasone did not induce inhibitory influences on the K pared from male Wistar rats weighing 200-250 g, and drug U50,488H, but it did induce a slight, although signi- immediately placed in a submerged recording chamber, where ficant potentiating effect. In the experiments performed with they were constantly perfused (at a rate of 2-3 ml min') a drug which has highly selective 62 effects (deltorphin II), with an artificial cerebrospinal fluid (CSF) saturated with dexamethasone had no effect (Table 2). The antagonistic 95% 02: 5% C02. The composition of the artificial CSF was effect of dexamethasone on DAMGO or morphine antino- 1418 S. PIERETTI et al. ciception was completely prevented by pretreatment with characterized by an increase of the amplitude of the CAl PS cycloheximide, a protein synthesis inhibitor (Table 2). from 2-5 to 7-11 mV, the appearance of some (1 to 3) additional population spikes and an increase in duration up In vitro experiments to 8-15 ms (Sagratella et al., 1987; Frank et al., 1988). These effects completely disappeared within 30-60 min after the Slice perfusion with 300 jLM morphine (n = 10) or 100 M drug was withdrawn from the perfusing solution (n = 6). methadone (n = 10) or 0.5 jtM DAMGO (n =6) or 10 pM Within 60 min of slice perfusion with 0.5JM of DPDPE morphiceptin (n = 6) in 100% of the experiments within (n = 5) or 0.5 gM of deltorphin II (n = 10) these drugs failed 60 min changed the CAl FP into an epileptiform bursting to produce the appearance of epileptiform bursting, but they induced only an increase of the amplitude of the CAl PS (PPSA= + 51 ± 11%). Slice perfusion with 150LM of the v. agonist, U50,488H (n = 6) induced a decrease of the amplitude of the CAl PS (PPSA= to 40.5 ± 7%) within 60 min. Dexamethasone perfusion (100 ltM, n = 4) for 60 min did not significantly affect the CAl FP amplitude (PPSA = + 12 + .a 6%) or duration (FPD= + 5 ± 3%). Dexamethasone per-

164( C Table 2 Effects of dexamethasone pretreatment (Dex, 0 0.1 mg kg-' administered 30 min before opioid agonist), a1) cycloheximide pretreatment (Cyclo, 15 mg kg-' administered CD 2 h before dexamethasone or 2.5 h before opioid agonist) on 40 the analgesia induced by i.c.v. injection of some opioid a1) agonists .40.- -6 0) a) Tail flick test Hot plate test .5 Drugs Mean ± s.e. Mean ± s.e. .0 Morphine 1.6 nmol/mouse 69.1 + 1.51] * 63.5 ± 1.7 1 ,* .0 Dex + morphine 43.5 ± 2.1 40.2 ± 1.7 0~ 4 Cyclo + morphine 63.0 ± 2.0 70.0 ± 1.5 Cyclo + Dex + morphine 72.3± 1.7 68.9 ± 2.5 DAMGO 0.05 nmol/mouse 65.0+ 3.1] * 53.0 ± 4.0 ] *, 0.01 0.1 1 10 100 Dex + DAMGO 33.6 ± 2.3 40.1 ± 1.7 Cyclo + DAMGO 68.9 ± 2.7 55.0 ± 1.4 Dose (nmol/mouse) Cyclo + Dex + DAMGO 70.0± 1.7 57.0 ± 2.0 Figure 1 Dose-response curves of various i.c.v. doses of some Deltorphin II opioid agonists alone or in the presence of dexamethasone pretreat- I nmol/mouse 50.0 ± 4.3 63.0 ± 2.1 ment (Dex 0.1 mg kg-' administered i.p. 30 min before opioid Dex + deltorphin II 43.0 ± 1.3 54.0 ± 4.0 agonist) in the tail flick (a) and in the hot plate tests (b). The latency of the responses was expressed as percentage of analgesia. This was Changes in the latency were expressed as percentage of calculated as (post-opioid latency - pre-drug latency)/(cut latency analgesia which was calculated as (post-opioid latency - - pre-drug latency) x 100, where the cut off time was 10 s in tail pre-drug latency)/(cut-off latency - pre-drug latency) x 100, flick and 60s in hot plate. In control animals the latency of the where the cut off time was 10 s in tail flick and 60 s in hot responses was 2-3 s in the tail flick test and 15- 17 s in the hot plate plate. Statistical analysis was performed by using the test. The ED50 and 95% confidence intervals were computed from Student's test. Groups consisted of at least 20 animals; each the dose-response curve by the method of Litchfield & Wilcoxon animal was tested once. (1949) with the aid of a computer programme. *P<0.05.

Table 1 Effects of dexamethasone pretreatment (Dex, 0.1 mg kg-' i.p.) on the analgesia induced by i.c.v. injection of some opioid agonists Tail flick test Hot plate test ED~v nmol/mouse ED50 nmol/mouse Drugs (95% confidence limits) (95% confidence limits)

Morphine 0.92 (0.73-1.17)] * 1.42 (1.13-1.78) ] * Dex + morphine 3.20 (2.65-3.86) 4.51 (3.66-5.56) P-Endorphin 0.10 (0.078-0.14)] * 0.080 (0.058-0.11) ] * Dex + P-endorphin 0.36 (0.24-0.53) 0.200 (0.144-0.27) DAMGO 0.04 (0.029-0.055) ] * 0.078 (0.056-0.10) ] * Dex+ DAMGO 0.66 (0.35-1.23) 0.18 (0.128-0.268) DPDPE 3.33 (2.45-4.52) ] * 3.44 (0.83-14.2) Dex + DPDPE 5.84 (3.99-8.56) 5.50 (4.15-7.29) U-50,488H 76.10 (63.2-91.6)] * 61.4 (50.4-74.7) Dex + U-50,488H 55.4 (46.6-65.8) 48.8 (22.6-105.5) Morphine (0.6-5.2 nmol), S-endorphin (0.015-0.6 nmol), DAMGO (0.01-0.2 nmol), DPDPE (0.5-4 nmol), U-50,488H (15-100 nmol) were administered 30 min after dexamethasone injection. Changes in the latency of the responses were expressed as percentage of analgesia which was calculated as (post-opioid latency - pre-drug latency)/(cut off latency - pre-drug latency) x 100, where the cut off time was 10 s in tail flick and 60 s in hot plate. The ED50 and 95% confidence intervals were computed from the dose-response curve by the method of Litchfield and Wilcoxon with the aid of the computer programme. Statistical analysis was carried out by Student's test. Groups consisted of at least 20 animals; each animal was tested only once. *Significant ratio (at least P<0.05). DEXAMETHASONE INTERFERENCE IN OPIOID EFFECTS 1419 fusion (100 riM) for 30 min followed by dexamethasone (100 gM) application plus DPDPE (0.5.uM, n = 6) or deltor- (100 ;M) plus morphine (300 gM) or methadone (100 gM) phin II (0.5 gM, n = 6) failed within 60 min to affect (n = 6) or DAMGO (0.5.M) (n = 6) or morphiceptin (10 atM) significantly the increase of the CAl PS amplitude due to the (n = 6) significantly reduced, within 60 min, the increase of 6 opioid agonists. the CAl FP duration and the number of additional popula- Dexamethasone perfusion (100 FM) for 30 min followed by tion spikes due to the p opiate agonists. Dexamethasone dexamethasone (100 gM), plus U50,488H (150 gM) (n = 6) perfusion (100 pM) for 30 min followed by dexamethasone failed within 60 min to affect significantly the decrease of the CAl PS amplitude due to the x opioid agonist. Slice perfusion for 30 min with the protein synthesis inhibitor, cycloheximide, at a concentration of 1 AM (n =4) 8 did not significantly change CAl FP amplitude (PPSA= 7- 6 5 a 60 min post Morphiceptin (10 FM) > 4 3 2

0 v At 14 12 5 mV 10 (A 8 5 ms E 6' 4 60 min post Morphiceptin (10 IM) + Dex (100 iM) 'after a 30 min pretreatment with 2 b Dex (100 gM) 0

c 3.0 2.5 10 X 2.0 8 1.5 I5mV E 1.0 z 0.5 5 ms 0.0 Morph Mor DAMGO Meth 60 min post Morphiceptin (10 gM) + Dex Figure 2 Influence of dexamethasone (Dex) on the electrophysio- (100 gM) + Cyc (1 gM) after a 30 min pretreatment logical effects of selected opioids on hippocampal CAl pyramidal c with Dex (100 FM) + Cyc (1 gM) neurones. (a) Effects on CAI PPSA: the histograms show the influence of Dex (100I1M) on opioid-induced effects on the amplitude of the CAI primary population spike amplitude (measured from the beginning to the maximum of the negative deflection of the extracellular field potential). (b) Effects on CAl FDP: the histograms show the influence of Dex (100 IsM) on opioid-induced effects on the duration of the CAl field potentials (measured from the first to the last population spike whose amplitude was higher than 0.2 mV). (c) M11IAAw Effects on CAl AEPS: the histograms show the influence of Dex V v mV (1001M) on opioid-induced occurrence of additional epileptiform population spikes. Abbreviations: DAMGO = 0.5 ILM DAMGO slice perfused for 60 min; Morph = 10 gIM morphiceptin slice perfused for 5 ms 60 min; METh = 100 gM methadone slice perfused for 60 min; Mor = 300 gM morphine slice perfused for 60 min; DPDPE = 0.5 I4M DPDPE slice perfused for 60 min Delt = 0.5 gIM deltorphin II slice perfused for 60 min; U-50 = 150SM U-50,488 H slice perfused Figure 3 Influence of dexamethasone and cycloheximide on mor- for 60 min; + Dex = 30 min slice perfusion with 100 gM dexametha- phiceptine epileptiform bursting in rat hippocampal slices (a) The sone followed by 60 min slice perfusion with I100 jM dexamethasone record shows the epileptiform bursting induced in rat hippocampal plus the opioid; + Cyc = 30 min slice perfusion with I jM cyclohex- slices within 60 min from the perfusion of IO M morphiceptin; (b) imide plus 100 jM dexamethasone followed by 60 min slice perfusion 30 min after dexamethasone (Dex, 1o0 AiM) pretreatment and 60 min with I gIM cycloheximide plus 100 jIM dexamethasone plus the opioid;' after perfusion of morphiceptin (10 iM), continuing the dexametha- * = significantly different from control (NONE, P<0.01 'according sone (100 jFM) perfusion, dexamethasone blocked the rise of epilepti- to Newman Keuls test); Significantly different from + Dex form bursting induced by morphiceptin; (c) 60 min after perfusion of (P<0.01 according to Newman Keuls test). Open columns: opioid; morphiceptin (10 M) continuing the dexamethasone (100IIM) plus hatched columns: dexamethasone plus opioid; cross-hatched col- cycloheximide (Cyc 1 jiM) perfusion, dexamethasone did not block umns: cycloheximide plus dexamethasone plus opioid. the rise of epileptiform bursting induced by morphiceptin. 1420 S. PIERETTI et al.

- 12 ±11%) or duration (FPD= +2 ± 8.6%). Adding discrepancy between in vivo and in vitro experiments might cycloheximide (1 fiM) (n = 6) for 30 min to the medium per- indicate that the influence of dexamethasone on K opioid- fusing the slice containing dexamethasone (1I00 M) followed induced analgesia did not involve the hippocampus. by a 60 min perfusion of morphine (300 JtM), methadone Various electrophysiological effects of corticosteroids on (1I00 IM) or DAMGO (0.5 fM) plus dexamethasone (100 pM) hippocampal neurones are described in the literature. Preva- completely blocked the previously described dexamethasone lent short-lasting neuronal depressant or membrane hyper- effects on the p agonists (Figures 2 and 3). polarising activity are described within a short time from hippocampal slice perfusion with cortisol succinate or corticosterone and, in agreement with our results, dexametha- Discussion sone perfusion resulted in few effects (Vidal et al., 1986; Joels & De Kloet, 1989; Chen et al., 1991). Reports in the liter- These results confirm and extend previous findings that dex- ature support the view that effects mediated by steroids amethasone induces inhibition of two main effects related to involve intracellular mechanisms on the genome. Recently it the opioid system, i.e., analgesia and hippocampal excit- has been shown that a high corticosterone concentration and ability. Moreover, this inhibition is quite evident as an the selective glucocorticoid RU 28362 1 h after application antagonism of the 1A opioid receptor, as tested with the tail increase the amplitude of the afterhyperpolarisation of CAl flick and hot plate antinociception tests, and with the in vitro pyramidal neurones obtained from adrenalectomized rats re- hippocampal model. sulting in a potential suppression of hippocampal excitability The different influence of dexamethasone on opioid-induc- (Kerr et al., 1989; Joels & de Kloet, 1989). This effect did not ed effects is probably dependent on the different mechanisms occur in the presence of the protein synthesis inhibitor, cyclo- that are activated after It and 6 opioid receptor stimulation. heximide, thus suggesting a genomic action of steroids (Karst We should note that the inhibition exerted by dexamethasone & Joels, 1991). Furthermore, it was reported that high levels on the ft opioid agonist is strong and highly reproducible. of corticosterone also affect calcium action potentials other However the inhibition exerted by dexamethasone on effects than the slow afterhyperpolarisation (Kerr et al., 1989). Our induced by the highly specific 62 agonist, deltorphin II was results indicate that at least 15-30 min latency is necessary not significant in both the tail flick and hot plate tests. The for the manifestation of the inhibitory properties exerted by inhibition exerted by dexamethasone on the 6, agonist, dexamethasone on these opioid effects, and confirm also that DPDPE, was of borderline significance in the tail flick test these effects are prevented by pretreatment with the protein and was not consistent in the hot plate test. Both DPDPE synthesis inhibitor, cycloheximide. All these findings suggest and deltorphin II in contrast to ft opioids failed to induce that the inhibitory effects that glucocorticoids exert on hippo- epileptiform bursting in the rat hippocampus model. The campal excitability (Joels & de Kloet, 1992) via protein- inability of the selective 6 agonists to induce spontaneous or synthesis mechanism may explain dexamethasone antagonism evoked epileptiform burstings, which was also shown in the on opioid-induced effects. This antagonism has been repro- dentate gyrus, is probably dependent on the inability of 6 duced without substantial modifications using two in vivo agonists to affect hippocampal recurrent inhibition (Lupica et models for the evaluation of the analgesic effects, and one in al., 1992). However, g and 6 agonists also affected hippocam- vitro model for the evaluation of cerebral excitability. In our pal non-recurrent synaptic inhibition, an effect revealed in previous paper (Pieretti et al., 1992) data very similar to extracellular studies by an increase of the CAl PS amplitude. those obtained in vitro (i.e., strong inhibition induced by Dexamethasone was not able to prevent it. Since hippocam- dexamethasone on the epileptiform effects elicited by mor- pal recurrent inhibition depends on the activity of GABAer- phine) were obtained also in vivo in rabbits treated i.v. with gic interneurones (Neumaier et al., 1988), the specific dexamethasone followed by morphine injected i.c.v. On that influence of dexamethasone on opioid-induced epileptiform occasion, deamethasone was also able to prevent consistently bursting (but not on the opioid-induced increase of the amp- EEG background alterations as well as gross behavioural litude of the CAl PS) might indicate a possible influence of alterations induced by morphine. Therefore, there is sufficient dexamethasone on GABA-mediated opioid-induced effects. evidence to hypothesize that the interference exerted by dex- In agreement with this hypothesis, in preliminary experiments amethasone on some opioid effects depends on the capacity we noted that dexamethasone was able to prevent of dexamethasone to affect the n-mediated effects throughout significantly the CAl epileptiform bursting due to the GABA a mechanism involving protein synthesis. Recently Magnuson antagonists, penicillin but not due to 'magnesium free' solu- et al. (1990) investigated the electrophysiological action of tions (Sagratella, unpublished observations). cholecystokinin octapeptide on the reduction of the C-fibre Dexamethasone also induced a slight, but significant evoked dorsal horn neurone activity elicited by trans- potentiation of the U50,488H-induced analgesia measured cutaneous electrical stimulation induced by DAMGO and with the tail flick test, and a significant potentiation on the DSTBULET (Tyr-D-Ser(otbu)-Gly-Phe-Leu-Thr), a 6 opioid hot plate test. The potentiating effect of dexamethasone on agonist. Magnuson reported that cholecystokinin selectively the analgesic effects of U50,488H is not surprising. In fact, prevented the inhibition of C-fibre evoked activity induced by opposite interactions between IL and K receptors have been DAMGO whereas no effect was shown on the inhibition of reported in different assays of analgesia (Ramarao et al., C-fibre evoked activity induced by DSTBULET. Others 1988; Craft & Dykstra, 1992) and discriminative stimulus reported that cholecystokinin octapeptide antagonized the effects (Negus et al., 1991). In fact, in the rat hippocampal analgesia mediated by the 1t agonist, PLO17 or morphine slice preparation, the K opioid U50,488H by itself in contrast (Faris et al., 1983) whereas the peptide did not change the to p and 6 agonists induced a consistent diminution in analgesic effects induced by the 6 agonist DPDPE (Wang et amplitude of CAl PS in rat hippocampal slices. This effect al., 1990). Studies performed in vivo have shown that stress was not modified by dexamethasone pretreatment in in vitro induced changes in cholecystokinin concentration in the rat experiments. Previous studies have also shown that K agon- hypothalamus, and that dexamethasone treatment increased ists such as dynorphin produced depressive effects on hippo- the cellular content and secretion of some cholecystokinin campal synaptic transmission in vivo and in vitro experiments peptides in a thyroid carcinoma cell line (Odum & Rehfeld, (Brookes & Bradley, 1984; Moises & Walker, 1985; Iwama et 1990). Dexamethasone treatment was also reported to be able al., 1986). Recent studies also demonstrated that other high to triple the number of cholecystokinin receptors per cell affinity K agonists such as U-69593 and U-54494A showed with little change in receptor affinity, and it was shown that depressant effects on the electrical synaptic response at the dexamethasone increased the sensitivity of AR42J pancreatic CAl and CA3 hippocampal areas (Alzheimer & Bruggencate, acinar cells to cholecystokinin (Logsdon, 1986). The possi- 1990; Proietti et al., 1991) and the discussion on the possible bility that dexamethasone exerts its antagonism versus opioid nature of this phenomenon is still open. However, the opioid-induced effects via cholecystokinin may be considered, DEXAMETHASONE INTERFERENCE IN OPIOID EFFECTS 1421 although some reports indicate that cholecystokinin may also This research wag supported in part by grants from Consiglio reduce K-induced analgesia (Wang et al., 1990) or selectively Nazionale delle Richerche (CNR N:9301752). We thank Ms Susan antagonize only P-endorphin-induced analgesia (Itoh et al., Holt for the English revision of the manuscript. 1982; Tseng & Collins, 1991). Finally, whatever the mechanism may be, our data indicated that glucocorticoid and opioid may exert inter-regulatory properties on pain and cerebral excitability.

References ALZHEIMER, C. & BRUGGENCATE, G.T. (1990). Non-opioid actions MACLENNAN, A.J., DRUGAN, C.R., HYSON, R.L., MAISER, S.F., of the k-opioid receptor agonist U50488H and U69593 on the MADDEN, IV, J. & BARCHAS, J.D. (1982). Corticosterone: a electrophysiologic properties of hippocampal CA3 neurons in critical factor in an opioid form of stress-induced analgesia. vitro. J. Pharmacol. Exp. Ther., 255, 900-905. Science, 215, 1530-1532. AMIR, S., BROWN, Z.W. & AMIT, Z. (1980). The role of endorphins in MAGNUSON, D.S.K., SULLIVAN, A.F., SIMONNET, G., ROQUES, B.P. stress: evidence and speculations. Neurosci. Behav. Rev., 4, & DICKENSON, A.H. (1990). Differential interactions of 77-87. cholecystokinin and FLFQPQRF-NH2 with 1s and 6 opioid anti- BROOKES, A. & BRADLEY, P.B. (1984). Electrophysiological evidence nociception in the rat spinal cord. Neuropeptides, 16, 213- for a k-agonist activity of dynorphin in rat brain. Neuropharma- 218. cology, 23, 207-210. McEVEN, B.S., DE KLOET, E.R. & ROSTENE, W. (1986). Adrenal CAPASSO, A., DI GIANNUARIO, A., LOIZZO, A., PIERETTI, S. & steroid receptors and actions in the nervous system. Physiol. Rev., SORRENTINO, L. (1992). Central interaction of dexamethasone 66, 1121-1188. and RU-38486 on morphine antinociception in mice. Life Sci., MILLAN, J.M. (1986). Multiple opioid system and pain. Pain, 27, 51, 139-143. 303-347. CHATTERJEE, T.K., SUMANTRA, D., BANERJE, P. & GHOSH, J.J. MOISES, H.C. & WALKER, M.J. (1985). Electrophysiological effects of (1982). Possible physiological role of adrenal and gonadal dynorphin peptides on hippocampal pyramidal cells in rats. Eur. steroids in morphine analgesia. Eur. J. Pharmacol., 77, 119- J. Pharmacol., 108, 85-88. 121. NEGUS, S.S., PICKER, M.J. & DYKSTRA, L.A. (1991). Interaction CHEN, Y.Z., HUA, S.Y., WANG, C.A., WU, L.G., GU, Q. & XING, B.R. between the discriminative stimulus effects of mu and kappa (1991). An electrophysiological study on the membrane receptor- opioid agonist in the squirrel monkey. J. Pharmacol. Exp. Ther., mediated action of glucocorticoids in mammalian neurons. 256, 149-158. Neuroendocrinology, 53, 25-30. NEUMAIER, J.F., MAILHEAU, S. & CHAVIKIN, C. (1988). Opioid CRAFT, R.M. & DYKSTRA, L.A. (1992). Agonist and antagonist receptor-mediated responses in the dentate gyrus and CAl region activity of kappa opioids in the squirrel monkey: antinociception of the rat hippocampus. J. Pharmacol. Exp. Ther., 244, and urine output. J. Pharmacol. Exp. Ther., 260, 327-333. 564-570. FARIS, P.L., KOMISARUK, B.R., WATKINS, L.R. & MAYER, D.J. ODUM, L. & REHFELD, J.F. (1990). Expression and processing of (1983). Evidence for the neuropeptide cholecystokinin as an procholecystokinin in a rat medullary thyroid carcinoma cell line. antagonist of opiate analgesia. Science, 219, 310-312. Biochem. J., 271, 31-36. FRANK, C., SAGRATELLA, S., BENEDETTI, M. & SCOTTI DE CARO- ORTOLANI, E., DI GIANNUARIO, A., NEROZZI, D., ZAPPONI, G.A. & LIS, A. (1988). Comparative influence of calcium blocker and LOIZZO, A. (1990). Some endorphin derivatives and hydrocorti- purinergic drugs on epileptiform bursting in rat hippocampal sone prevent EEG limbic seizures induced by corticotropin- slices. Brain Res., 441, 393-397. releasing factor in rabbits. Epilepsia, 31, 702-707. HALEY, T.J. & McCORMICK, W.G. (1957). Pharmacological effect PASTERNAK, G.W. (1987). Opioid receptors. In Psychopharmacology: produced by intracerebral injections of drugs in the conscious the Third Generation of Progress. ed. Meltzer, H.Y. pp. 281-288. mouse. Br. J. Pharmacol. Chemother., 12, 12-15. New York: Raven Press. HONG, J.S., MCGINTY, J.F., LEE, P.H.K., XIE, C.W. & MICTHELL, C.L. PIERETTI, S., CAPASSO, A., DI GIANNUARIO, A., LOIZZO, A. & (1993). Relationship between hippocampal opioid peptides and SORRENTINO, L. (1991). The interaction of peripherically and seizures. Progr. Neurobiol., 40, 507-528. centrally administered dexamethasone and RU 38486 on mor- ITOH, S., KATSUURA, G. & MAEDA, Y. (1982). Caerulein and chole- phine analgesia in mice. Gen. Pharmacol., 22, 929-933. cystokinin suppress P-endorphin-induced analgesia in the rat. PIERETTI, S., DI GIANNUARIO, A., LOIZZO, A., SAGRATELLA, S., Eur. J. Pharmacol., 80, 421-425. SCOTTI DE CAROLIS, A., CAPASSO, A. & SORRENTINO, L. IWAMA, T., ISHIHARA, K., SATOH, M. & TAKAGI, H. (1986). Differ- (1992). Dexamethasone prevents epileptiform activity induced by ent effects of dynorphin on in vitro guinea pig hippocampal CA3 morphine in in vivo and in vitro experiments. J. Pharmacol. Exp. pyramidal cells with various degrees of paired-pulse facilitation. Ther., 263, 830-839. Neurosci. Lett., 63, 190-194. PROIETTI, M.L., SCOTTI DE CAROLIS, A., FRANK, C., ZENG, Y.C. & JOELS, M. & DE KLOET, E.R. (1989). Effects of glucocorticoids and SAGRATELLA, S. (1991). In vitro depressant effects of U-54494A, norepinephrine on the excitability in the hippocampus. Science, an anticonvulsant related to kappa opioids in the hippocampus. 245, 1502-1505. Neuropharmacol., 30, 637-642. JOELS, M. & DE KLOET, E.R. (1992). Control of neuronal excitability RAMABADRAN, K. & BANSINATH, M. (1990). Endogenous opioid by corticosteroid hormones. Trends Neurosci., 15, 25- 30. peptides and epilepsy. Int. J. Clin. Pharmacol. Ther. Toxicol., 28, KARST, H. & JOELS, M. (1991). The induction of corticosteroid 47-62. actions on membrane properties of hippocampal CAI neurons RAMARAO, P., JABLONSKI, H.I., REHDER, K.R. & BHARGAVA, H.N. requires protein synthesis. Neurosci. Lett., 130, 27-31. (1988). Effect of k-opioid receptor agonists on morphine anal- KERR, D.S., CAMPBELL, L.W., HAO, S.-Y., LANDFIELD, P.W. (1989). gesia in morphine-naive and morphine-tolerant rats. Eur. J. Phar- Corticosteroid modulation of hippocampal potentials: increased macol., 156, 239-246. effects with aging. Science, 245, 1505-1509. SAGRATELLA, S., FRANK, C. & SCOTTI DE CAROLIS, A. (1987). LEWIS, J.W., CANNON, J.T. & LEIBESKIND, J.C. (1980). Opioid and Effects of ketamine and (+) cyclazocine on 4-aminopyridine and non-opioid mechanisms of stress analgesia. Science, 208, 623- 'magnesium free' epileptogenic activity in hippocampal slices of 625. rats. Neuropharmacology, 26, 1181-1184. LITCHFIELD, J.T. & WILCOXON, F. (1949). A simplified method of SPIESS, J., RIVIER, J., RIVIER, C. & VALE, W. (1981). Primary struc- evaluating dose effects experiments. J. Pharmacol. Exp. Ther., 96, ture of corticotropin releasing factor from ovine hypothalamus. 99-113. Proc. Nat!. Acad. Sci. U.S.A., 78, 6517-6521. LOGSDON, C.D. (1986). Glucocorticoids increase cholecystokinin TSENG, L.F. & COLLINS, K.A. (1991). Cholecystokinin administered receptors and amylase secretion in pancreatic acinar AR42J cells. intrathecally selectively antagonizes intracerebroventricular P- J. Biol. Chem., 261, 2096-2101 endorphin-induced tail flick inhibition in the mouse. J. Phar- LUPICA, C.R., PROCTOR, W.R. & DUNWIDDLE, T.V. (1992). Dis- macol. Exp. Ther., 260, 1086-1092. sociation of ft and 6 opioid receptor-mediated reduction in evoked and spontaneous synaptic inhibition in the rat hippocampus in vitro. Brain Res., 593, 226-238. 1422 S. PIERETTI et al.

VALE, W., SPIESS, J., RIVIER, C., RIVIER, J. (1981). Characterization WINTER, C.A. & FLATAKER, J. (1951). The effect of corticosterone, of a 41-residue ovine hypothalamic peptide that stimulates the desoxycorticosterone and adrenocorticotropic hormone upon the secretion of corticotropin and b-endorphin. Science, 231, 1394- response of animals to analgesic drugs. J. Pharmacol. Exp. Ther., 1397. 103, 93-95. VIDAL, C., JORDAN, W. & ZIEGLAGANSBERGER, W. (1986). Corti- ZIMMERMAN, M. (1983). Ethical guidelines for investigations of costerone reduces the excitability of hippocampal pyramidal cells experimental pain in conscious animals. Pain, 16, 109-110. in vitro. Brain Res., 383, 54-59. WANG, X.-J., WANG, X.-H. & HAN, J.-S. (1990). Cholecystokinin (Received April 12, 1994 octapeptide antagonized opioid analgesia mediated by pl- and K- Revised June 24, 1994 but not 5-receptors in the spinal cord of the rat. Brain Res., 523, Accepted July 25, 1994) 5-10.