Ultra-low dose naltrexone potentiates the anticonvulsant effect of low dose morphine on clonic seizures

Neuroscience 129 (2004) 733–742

ULTRA-LOW DOSE NALTREXONE POTENTIATES THE ANTICONVULSANT EFFECT OF LOW DOSE MORPHINE ON CLONIC SEIZURES H. HONAR,a K. RIAZI,a H. HOMAYOUN,a H. SADEGHIPOUR,a N. RASHIDI,b M. R. EBRAHIMKHANI,a N. MIRAZIc AND A. R. DEHPOURa*

triphosphate-binding protein and effectors such as adenylate cyclase and ion channels (Childers, 1991; Kieffer, 1995). Opioids exert a wide range of their effects, including their analgesic properties, through coupling to inhibitory Gi/Go proteins, leading to decreased neuronal cyclic AMP levels, decreased Ca⫹⫹ conductance, shortening of action potential duration (APD) and decreased neurotransmitter release (Williams et al., 2001, for review). However, some recent data suggest that opioids may also have direct stimulatory effects on intracellular signaling mechanisms including stimulation of adenylyl cyclase, increasing calcium influx, prolongation of APD and increased neuronal excitability (Chen et al., 1988; Shen and Crain, 1989; Crain and Shen, 1990; Gintzler and Xu, 1991; Cruciani et al., 1993; Mao et al., 1994; Smart and Lambert, 1996). According to Crain and colleagues (Shen and Crain 1997; Crain and Shen, 1995, 2000) very low doses of opioids can selectively activate an excitatory Gs proteincoupled signaling pathway that may account for the mentioned stimulatory effects of opioids. It has been suggested that ultra-low doses of an opioid receptor antagonist can selectively block this sensitive excitatory mechanisms and unmask a potent inhibitory tone that is induced by opioids at concentrations much lower than their minimal effective doses in antinociception tests. Accordingly, evidence has been provided by several basic and clinical groups in favor of the significant potentiation of opioid-induced antinociception by ultra-low doses of opioid receptor antagonists (Levine et al., 1988; Crain and Shen, 1995; Gan et al., 1997; Powell et al., 2002; Cruciani et al., 2003). These putative excitatory mechanisms may also be responsible, at least partly, for the “paradoxical” phenomenon of opioidinduced hyperalgesia that is shown to be reversible by ultra-low doses of opioid antagonists (Wiesenfeld-Hallin et al., 1991; Crain and Shen, 2001). In addition, pretreatment with ultra-low doses of opioid receptor antagonists can inhibit the development of tolerance to morphine-induced analgesia. This effect is suggested to be through blockade of a switch from early dominant inhibitory to later dominant excitatory signaling by chronic opioid treatment (Powell et al., 2002; Low et al., 2003; Wang et al., 2003). While it is not certain to what extent the reported coupling of opioid receptors to Gs second messenger system in pain-related pathways may be observed in other neural tissues, the excitatory pathway activated by very low doses of opioids may be of functional significance beyond the antinociception paradigms. One important field for investigation would be the modulatory effects of opioids on seizure susceptibility. There is

a

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran

b

Department of Biology, School of Science, University of Tehran, Tehran, Iran

c Department of Biology, School of Science, Bu Ali Sina University, Hamadan, Iran

Abstract—Significant potentiation of analgesic effects of opioids can be achieved through selective blockade of their stimulatory effects on intracellular signaling pathways by ultra-low doses of opioid receptor antagonists. However, the generality and specificity of this interaction is not well understood. The bimodal modulation of pentylenetetrazoleinduced seizure threshold by opioids provide a model to assess the potential usefulness of this approach in seizure disorders and to examine the differential mechanisms involved in opioid anti- (morphine at 0.5–3 mg/kg) versus proconvulsant (20 –100 mg/kg) effects. Systemic administration of ultra-low doses of naltrexone (100 fg/kg–10 ng/kg) significantly potentiated the anticonvulsant effect of morphine at 0.5 mg/kg while higher degrees of opioid receptor antagonism blocked this effect. Moreover, inhibition of opioidinduced excitatory signaling by naltrexone (1 ng/kg) unmasked a strong anticonvulsant effect for very low doses of morphine (1 ng/kg–100 ␮g/kg), suggesting that a presumed inhibitory component of opioid receptor signaling can exert strong seizure-protective effects even at very low levels of opioid receptor activation. However, ultra-low dose naltrexone could not increase the maximal anticonvulsant effect of morphine (1–3 mg/kg), possibly due to a ceiling effect. The proconvulsant effects of morphine on seizure threshold were minimally altered by ultra-low doses of naltrexone while being completely blocked by a higher dose (1 mg/kg) of the antagonist. The present data suggest that ultra-low doses of opioid receptor antagonists may provide a potent strategy to modulate seizure susceptibility, especially in conjunction with very low doses of opioids. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: excitatory opioid receptor, ultra-low dose naltrexone, pentylenetetrazole, clonic seizure threshold, mice.

Receptors of opioid peptides belong to the super family of G-protein-coupled receptors. These receptors exert their diverse effects through activation of guanosine *Corresponding author. Tel: ⫹98-21-611-2802; fax: ⫹98-21-640-2569. E-mail address: [email protected] (A. R. Dehpour). Abbreviations: APD, action potential duration; NTX, naltrexone; PTZ, pentylenetetrazole.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.08.029

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Fig. 1. Effect of different doses of morphine on PTZ-induced seizure threshold. Morphine (0.000001, 0.00001, 0.001, 0.1, 0.5, 1, 3, 7.5, 15, 20, 30, 60 and 100 mg/kg, s.c.) was administered 15 min after an i.p. injection of saline (vehicle instead of NTX) and 45 min before determination of PTZ seizure threshold. Data are expressed as mean⫾S.E.M. of seizure threshold in each group. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 compared with saline/saline control group.

considerable evidence supporting a protective role for endogenous opioids against several distinct models of epileptic disorders (Frenk, 1983; Lauretti et al., 1994; Khavandgar et al., 2003), in post-ictal period (Lee and Lomax, 1984; Molaie and Kadzielawa, 1989), and in association with stress (De Lima and Rae, 1991; Homayoun et al., 2002a). Although exogenous opioid receptor agonists like morphine (at 0.5–5 mg/kg) can simulate such anticonvulsant effects, their potential clinical application in epilepsy is hindered by their potent side effects at this dose range. Since the anticonvulsant effect of opioids is suspected to be mediated through inhibitory G proteins (Lauretti et al., 1994), we hypothesized that 1) blocking the sensitive excitatory effects of opioid receptors by ultra-low doses of naltrexone (NTX) may allow potentiation of the inhibitory component of opioid receptor signaling and the associated anticonvulsant effect. To address this question, we assessed the clonic seizure threshold induced by i.v. administration of GABA receptor antagonist pentylenetetrazole (PTZ). Furthermore, based on the obtained results and previous literature on antinociception, we hypothesized that 2) combination of ultra low doses of NTX with very low and non-effective doses of morphine may produce an anticonvulsant effect. It should be noted that PTZ exerts its convulsant effect through specific interaction with the GABAA-gated chloride ionophore, resulting in the hyperexcitability of epileptogenic centers in CNS. This paradigm represents an animal model of petit-mal myoclonic seizures and is very sensitive to changes in seizure susceptibility (Swinyard and Kupferberg, 1985; Löscher et al.,

1991). Moreover, this model is able to detect both the anticonvulsant effects of low dose opioids as well as a strong proconvulsant effect induced by the higher doses of opioids (morphine, 20 –100 mg/kg). The latter effect has clinical relevance for convulsive episodes encountered in patients medicated with high dose opioids. These proconvulsant properties are believed to involve widespread activation of inhibitory signaling pathways leading to generalized disinhibition of epileptic centers (Zieglgänsberger et al., 1979; Lauretti et al., 1994). Thus, we also 3) examined the effect of ultra low doses of NTX on the proconvulsant properties of morphine. Finally, we used another distinct and clinically relevant experimental model of seizure, namely latency for the onset and the incidence of the generalized tonic– clonic seizures after acute i.p. injection of a relatively high dose of PTZ (Löscher et al., 1991; Kupferberg, 2001), to examine 4) whether interaction between low doses of opioid receptor agonist and antagonist in modulation of seizure extends to this model of grand-mal seizures as well.

EXPERIMENTAL PROCEDURES Chemicals Drugs used were PTZ, morphine sulfate (Mor) and NTX (all purchased from Sigma, Poole, UK). Morphine sulfate and NTX were dissolved in physiologic saline solution to such concentrations that requisite doses were administered in a volume of 10 ml/kg of the mice body weight. PTZ was prepared in saline as 1% solution. In

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Fig. 2. Effect of different doses of NTX on PTZ-induced seizure threshold. Graded doses of NTX (1 fg–1 mg/kg, i.p.) were administered 15 min before s.c. injection of saline (vehicle instead of morphine) and 60 min before determination of PTZ seizure threshold. Data are expressed as mean⫾S.E.M. of seizure threshold in each group. NTX at any of doses used had no effect on PTZ seizure susceptibility.

all experiments morphine sulfate was administered s.c. and NTX was administered i.p.

Subjects Male Swiss mice (Razi Institute, Karaj, Iran) weighing 24 –29 g at the time of the experiments were used. The animals were housed in standard polycarbonate cages in a temperature-controlled room (24⫾2 °C) on a 12-h light/dark cycle with free access to food and water and were acclimated at least 2 days before experiments. The experiments were conducted between 9:00 AM and 3:00 PM and all procedures conformed to the international guidelines on the ethical use of animals. All possible measures were undertaken to minimize the number of animals used and also to minimize animal’s discomfort including immediate euthanasia after acute experiments. Each mouse was used only once and each treatment group consisted of at least seven animals.

Determination of seizure susceptibility Clonic seizure threshold. PTZ-induced clonic seizure threshold was determined by inserting a 30 gauge butterfly needle into the tail vein of mice and the infusion of PTZ (1.0%) at a constant rate of 1 ml/min to unrestrained freely moving animals. Infusion was halted when forelimb clonus followed by full clonus of the body was observed. Minimal dose of PTZ (mg/kg of mice weight) needed to induce clonic seizure was considered as an index of seizure threshold. Tonic– clonic generalized seizure. Acute i.p. administration of PTZ (85 mg/kg) was used to evaluate the incidence and the latency for the onset of generalized tonic– clonic seizures and the incidence of death following seizures (Löscher and Lehmann, 1996; Homayoun et al., 2002c; Shafaroodi et al., 2004). The time of observation following PTZ injection was limited to 30 min.

Treatment Experiment 1 examined the effects of a wide dose-range of morphine (1 and 10 ng/kg, 1 ␮g/kg, and 0.1, 0.5, 1, 3, 7.5, 15, 20, 30, 60 and 100 mg/kg) on clonic seizure threshold. Animals in this

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experiment received acute s.c. injections of saline or mentioned doses of morphine 45 min before determination of PTZ-induced clonic seizure threshold. For all the animals in this experiment an i.p. injection of saline (vehicle instead of NTX) was administered 15 min prior to morphine administration and 60 min before determination of seizure threshold. Experiment 2 examined the effects of a wide dose-range of NTX (1 fg/kg–1 mg/kg, i.p.) by itself on clonic seizure threshold. In this experiment, mice were treated with graded doses of NTX 15 min before s.c. administration of saline (vehicle instead of morphine) and 60 min prior to determination of PTZ seizure threshold. Experiment 3 examined whether the weak anticonvulsant effect of morphine at 0.5 mg/kg may be altered/potentiated by ultra-low doses of NTX. Mice in this experiment received acute injections of a wide-range of ultra-low doses of NTX (1, 10 and 100 fg/kg, 1 and 100 pg/kg, 1, 10 and 100 ng/kg, 100 ␮g/kg) 15 min before morphine (0.5 mg/kg) and 60 min prior to PTZ injection. For comparison, one group received 1 mg/kg of NTX before morphine. In experiment 4, we examined the hypothesis that ultra-low dose opioid receptor antagonist may unmask a potential anticonvulsant effect exerted by very low doses of morphine. Thus, an ultra-low dose of NTX (1 ng/kg, a dose showing strong potentiation of the anticonvulsant effect of morphine at 0.5 mg/kg) was administered 15 min before very low doses of morphine (100 pg/kg, 1 and 10 ng/kg, 1 and 100 ␮g/kg) and 60 min prior to determination of PTZ seizure threshold. Experiment 5 examined whether ultra-low dose NTX may induce supramaximal enhancement of the anticonvulsant effects of morphine. NTX (1 ng/kg, i.p.) was administered 15 min before doses of morphine (1 and 3 mg/kg) that induce maximal anticonvulsant effects and 60 min before PTZ. Experiment 6 examined the effects of ultra-low dose range of NTX (100 pg/kg–10 ng/kg) on the proconvulsant effects of the high doses of systemic morphine (20, 60 and 100 mg/kg) on clonic seizure threshold. For comparison, two groups of mice were pretreated with 1 mg/kg of NTX before receiving potent proconvulsant doses of morphine (60 and 100 mg/kg). In experiment 7, we examined the effect of concomitant administration of saline or an ultra-low dose of NTX (1 or 10 ng/kg, i.p., based on the results of experiment 3) with either of two different doses of morphine (0.5 and 1 mg/kg, s.c.) on the time latencies for the onset of tonic generalized seizure after i.p. injection of PTZ (85 mg/kg). NTX was administered 15 min before morphine and 60 min before PTZ injection. A higher dose of morphine (3 mg/kg) was administered to another group to control for the anticonvulsant effect of morphine in this model. The dosage and time intervals were chosen based on previously published studies (Lauretti et al., 1994; Shen and Crain, 1997; Homayoun et al., 2002b; Shafaroodi et al., 2004) and pilot experiments. Vehicle controls were used in all experiments. For each experimental series, same-session controls were used to avoid possible environmental or day-to-day variations.

Statistical analysis In case of seizure threshold experiments, data were expressed as means⫾S.E.M. and comparisons between groups were made using one-way analysis of variance followed by post hoc TukeyKramer multiple comparisons. In case of i.p. PTZ paradigm, time latencies for tonic seizure had a wide distribution range, which was due to the fact that non-occurrence of tonic seizure was recorded as maximum observation time (1800 s). Thus, data were expressed as medians (with 95% confidence intervals) and a non-parametric analysis based on median values (Mann-Whitney U test) was used. Incidences of tonic generalized seizures and death subsequent to i.p. PTZ administration were compared between different groups using Fisher’s exact test. In all experiments a P-value less than 0.05 was considered statistically significant.

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Fig. 3. Biphasic dose-response effects of graded doses of NTX on the weak anticonvulsant property of a fixed dose of morphine (0.5 mg/kg, s.c.). NTX (1 fg–1 mg/kg, i.p.) was administered 15 min before s.c. injection of morphine and 60 min before determination of PTZ seizure threshold. The group morphine 3 mg/kg has been shown just for comparison. Data are expressed as mean⫾S.E.M. of seizure threshold in each group. * P⬍0.05, *** P⬍0.001 compared with saline/saline control group. # P⬍0.05, ### P⬍0.001 compared with saline/morphine 0.5 mg/kg group.

RESULTS Effect of different doses of morphine on seizure threshold As shown in Fig. 1 morphine induced a bimodal modulation of clonic seizure threshold. Lower doses of morphine (1 and 10 ng/kg, 1 ␮g/kg, and 0.1) did not alter seizure threshold. Doses of morphine between 0.5 and 3 mg/kg significantly increased seizure threshold with the maximum anticonvulsant effect at 3 mg/kg (post hoc analysis, P⬍0.001). Doses of morphine higher than 15 mg/kg significantly decreased the seizure threshold in a dosedependent manner (P⬍0.001). NTX by itself does not have any effect on PTZ-induced seizure threshold As shown in Fig. 2 acute administration of different doses of NTX (1 fg/kg, 1 pg/kg, 1 ng/kg, 1 ␮g/kg or 1 mg/kg, i.p.) by its own had no effect on PTZ-induced clonic seizure threshold (F(5,44)⫽1.188, P⬎0.05). Thus, it is unlikely that ultra low doses of NTX directly affect the seizure-regulating neurocircuitry. Pretreatment with ultra-low doses of NTX enhances the anticonvulsant effect of morphine As shown in Fig. 3, NTX at a dose of 1 mg/kg completely

reversed the anticonvulsant effect of morphine (0.5 and 3 mg/kg, s.c.). This effect is in agreement with the potent antagonistic action of NTX on opioid receptor functions and is similar to what has been previously reported for this dose of opioid receptor antagonist. Administration of a range of 10 –10,000-fold lower doses of NTX (100 ng– 100 ␮g/kg) did not have any significant effect on the anticonvulsant property of morphine at 0.5 mg/kg. By contrast, co-treatment of mice with the same dose of morphine (0.5 mg/kg) plus a 50,000 –5,000,000,000-fold lower dose of NTX (100 fg–10 ng/kg, i.p.) led to significant potentiation of the anticonvulsant effect of morphine (F(11,92)⫽16.342, P⬍0.001). The maximum anticonvulsant effect was observed with the doses of NTX between 100 pg/kg and 10 ng/kg (post hoc analysis, P⬍0.001). It is noteworthy that even pretreatment with doses as low as 1–10 fg/kg of NTX could mildly, though not significantly, increase the anticonvulsant effect of 0.5 mg/kg of morphine. Pretreatment with an ultra-low dose of NTX unmasks an anticonvulsant effect induced by very low doses of morphine As shown in Fig. 4a, the very low doses of morphine (100 pg–100 ␮g/kg) per se did not alter clonic seizure threshold. However, pretreatment with an ultra-low dose of NTX (1 ng/kg) unmasked significant anticonvulsant effects for the

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Pretreatment with ultra-low dose NTX does not increase the maximal anticonvulsant effect of morphine We further investigated the possibility of the potentiation of the maximal anticonvulsant effect of morphine by concomitant administration of ultra low dose NTX. Fig. 4b illustrates that the maximal anticonvulsant effects of morphine (at 1 or 3 mg/kg) were not augmented following pretreatment with an ultra-low dose of NTX (1 ng/kg, i.p.) (F(7,60)⫽23.871, P⬍0.001, post hoc analysis, P⬎0.05). Since the same pretreatment significantly potentiated the submaximal anticonvulsant effect of 0.5 mg/kg of morphine, the observed lack of effect may be due to a ceiling effect for this effect of morphine. Ultra-low doses of NTX did not alter the proconvulsant effects of the higher doses of morphine As demonstrated in Fig. 5, the higher doses of morphine (20, 60 and 100 mg/kg, s.c.) induced potent proconvulsant effects (F(12,99)⫽19.540, P⬍0.001). As previously reported, these effects are completely reversible by opioid receptor antagonists at the dose of 1 mg/kg (post hoc analysis, P⬍0.001). Pretreatment of mice with acute ultra-low doses of NTX (0.1, 1 or 10 ng/kg, i.p.) had no significant effect on the proconvulsant effects of the higher doses of morphine (post hoc analysis, P⬎0.05). It should be noted that doses of 1 ng/kg and 10 ng/kg of NTX mildly decreased the weak proconvulsant effect of morphine at 20 mg/kg such that their combination failed to show significant difference from saline/saline control group. However, the effect of pretreatment with ultra-low doses of NTX was not significantly different from that of the vehicle-pretreatment in any of the groups treated with proconvulsant doses of morphine (post hoc analysis, P⬎0.05). Effects of concomitant administration of ultra-low dose NTX and morphine on tonic generalized seizure latency and incidence Fig. 4. Pretreatment of mice with a fixed ultra-low dose of NTX (1 ng/kg, i.p.) unmasks the potent anticonvulsant effects of per se noneffective low doses (100 pg–100 ␮g/kg) of morphine (a), but has no significant effect on the maximal anticonvulsant properties of morphine’s regular doses (1 and 3 mg/kg, s.c.) (b). Saline or NTX was administered 15 min before s.c. injection of morphine and 60 min before determination of PTZ seizure threshold. Data are expressed as mean⫾S.E.M. of seizure threshold in each group. * P⬍0.05, *** P⬍0.001 compared with saline/saline control group. ### P⬍0.001 compared with corresponding saline/morphine group.

above dose range (100 pg–100 ␮g/kg) of morphine (F(11,94)⫽21.145, P⬍0.001). Even such a low dose of morphine as one ng/kg, when combined with ultra-low dose NTX, showed significant difference from saline/saline control group (post hoc analysis, P⬍0.05). The higher doses of morphine (10 ng–100 ␮g/kg) showed a significant additive anticonvulsant effect with ultra-low dose NTX (post hoc analysis, P⬍0.001).

The final experiment described in this study used another model of seizure (tonic– clonic) to examined whether the interaction between ultra low dose NTX and morphine would extend to this model. Table 1 shows that morphine at 3 mg/kg significantly increased the tonic seizure latency (Mann-Whitney U test, P⬍0.001) and decreased the incidences of both tonic seizure (Fisher’s exact test, P⬍0.05) and death (P⬍0.01) compared with saline control group. Lower doses of morphine (0.5 and 1 mg/kg) did not significantly change the tonic seizure latency (P⬎0.05) or the incidences of tonic seizures and death (P⬎0.05). NTX by itself did not have any effect on tonic seizure latency and incidence. We then examined the effect of concomitant administration of an ultra-low dose of NTX (1 or 10 ng/kg, i.p.) with either of two weak anticonvulsant doses of morphine (0.5 and 1 mg/kg, s.c.) in this model. Co-treatment of mice with an ultra-low dose of NTX (10 ng/kg) plus

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Fig. 5. Effect of ultra-low doses of NTX on proconvulsant activity of higher doses of morphine. Saline or NTX (0.1, 1 and 10 ng/kg, i.p.) was administered 15 min before s.c. injection of morphine (20, 60 and 100 mg/kg) and 60 min before seizure threshold determination. As illustrated above ultra-low dose NTX had no effect on proconvulsant properties of higher doses of morphine. Data are expressed as mean⫾S.E.M. of seizure threshold in each group. * P⬍0.05, *** P⬍0.001 compared with saline/saline control group. ### P⬍0.001 compared with corresponding saline/morphine group.

1 mg/kg of morphine increased the latency of tonic– clonic seizures to a level that was significantly higher than that of saline control group (Mann-Whitney U test, P⬍0.05). Also the incidence of tonic seizure (but not the death incidence) was significantly lower (Fisher’s exact test, P⬍0.05) compared with the same control group. NTX at a 10-fold lower dose (1 ng/kg), when co-administered with morphine (0.5 or 1 mg/kg), mildly increased the tonic seizure latency but this effect failed to show any statistical significance in comparison with saline control group.

DISCUSSION The present study provides evidence that opioid receptor antagonism by extremely low doses of NTX can produce a seemingly paradoxical effect on modulation of seizure susceptibility by opioids. We report that ultra-low doses of NTX can unmask a strong anticonvulsant effect induced by a very low dose range of systemic morphine (ng/kg–␮g/kg), and significantly potentiate its anticonvulsant effect at a 100 –100,000-fold higher dose (0.5 mg/kg). On the other hand, the proconvulsant effect of a higher dose range of

Table 1. Effects of ultra-low dose NTX and morphine on tonic generalized seizure latency and incidencea Treatment

N

Tonic seizure latency

TSI

DI

Saline⫹saline NTX (1 ng/kg)⫹saline NTX (10 ng/kg)⫹saline Saline⫹Mor (0.5 mg/kg) Saline⫹Mor (1 mg/kg) Saline⫹Mor (3 mg/kg) NTX (1 ng/kg)⫹Mor (0.5 mg/kg) NTX (1 ng/kg)⫹Mor (1 mg/kg) NTX (10 ng/kg)⫹Mor (1 mg/kg)

15 15 13 16 11 11 16 12 14

331 (259.5, 741.2) 386 (280.1, 867.1) 390 (206.3, 748.3) 472 (447.8, 1056.9) 446 (372.4, 1208.6) 1335 (997.6, 1672.6)a 654 (483.5, 1227.9) 628 (394.4, 1230.2) 726.5 (606.7, 1439.4)b

14 13 12 13 9 6b 11 10 8b

13 13 12 12 9 3c 11 9 8

a The latency to the onset of tonic generalized seizures as well as the incidence of tonic generalized seizures (TSI) and death (DI) was assessed following an 85 mg/kg PTZ i.p. injection. Saline or an ultra-low dose of NTX (1 or 10 ng/kg, i.p.) was administered 15 min before s.c. injection of saline or different doses of morphine and 75 min before i.p. injection of PTZ. Seizure latencies are expressed as medians (with 95% confidence intervals) in each group. The saline/morphine (3 mg/kg) has been shown just for comparison. a P ⬍ 0.001, b P ⬍ 0.05, c P ⬍ 0.01 compared to saline/saline control group.

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morphine (20 –100 mg/kg) was not significantly altered by such ultra-low dose antagonist pretreatment. The paradoxical potentiation of opioid-induced analgesia by ultra-low doses of opioid receptor antagonists have been previously described in several antinociception paradigms in experimental animals (Crain and Shen, 1995, 1998, 2001; Powell et al., 2002) as well as in some preliminary clinical reports (Levine et al., 1988; Gan et al., 1997; Cruciani et al., 2003). This phenomenon has been linked to the reported stimulatory effects of opioids at very low doses that include prolongation of action potential, increase in intracellular calcium release and protein kinase C-mediated signaling (Smart et al., 1994; Crain and Shen, 2000; Xu et al., 2003). The extent and functional importance of these observations are not currently known, though some researchers have provided evidence that these stimulatory effects may be important in opioid-induced hyperalgesia and development of tolerance and dependence to opioids (WiesenfeldHallin et al., 1991; Shen and Crain, 2001; Mao, 2002; Xu et al., 2003). Crain and colleagues have suggested that a specific kind of opioid receptors that is coupled to Gs second messenger proteins may mediate the stimulatory effects of the very low doses of opioids in dorsal root ganglion cells (Crain and Shen, 1990, 1995, 1998). While the coupling of opioid receptors to Gs proteins remains a controversial issue (Smart and Lambert, 1996; Connor and Christie, 1999), other researchers have reported some stimulatory effects for opioid receptors following coupling to Gi/Go proteins that also mediate their better known inhibitory effects (Olianas and Onali, 1994; Tsu et al., 1995; Kaneko et al., 1994). Regardless of the second messengers involved, the existence of an opioid receptor-activated stimulatory pathway that is often masked by the more classically recognized inhibitory pathway, raises the interesting possibility of modulating opioid-mediated functions by ultra-low doses of opioid antagonists (Crain and Shen, 1995, 2000; Powell et al., 2002; Oxbro et al., 2003; Wang et al., 2003). The PTZ-induced clonic seizures primarily model a type of forebrain-regulated seizures that are associated with increased activity in major epileptogenic centers of forebrain like amygdala and piriform cortex (Gale, 1992; Sarkisian, 2001; Eells et al., 2004). This type of seizure is subject to naloxone-reversible biphasic modulation by opioids (Lauretti et al., 1994; Homayoun et al., 2002b). The dose of opioid antagonist that blocks these effects is near 1 mg/kg that is much higher than the present ultra-low doses but still specific for ␮-opioid receptors. The anticonvulsant effect of opioids is thought to be mediated through inhibitory effects on excitatory pathways and increasing GABAergic tone (Sagratella and Massotti, 1982; Manocha et al., 2003). The potentiation of the significant but submaximal anticonvulsant effect of morphine at 0.5 mg/kg by ultra-low doses of NTX suggests that seizure-modulating effects of the low anticonvulsant doses of morphine may also consist of a sensitive excitatory component (that would be blocked by ultra-low doses of

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antagonist) as well as a less sensitive inhibitory component (that can be unmasked at very low doses of morphine). This profile of effects is similar to the effects of ultra-low doses of opioid antagonists on antinociception and analgesic tolerance (Crain and Shen, 1998; Powell et al., 2002; Low et al., 2003; Wang et al., 2003). It also resembles the dual excitatory/stimulatory mechanisms reported for low doses of opioids in some tissues including spinal cord and ileum (Gintzler et al., 1991; Rusin and Randic, 1991; Crain and Shen, 1995, 2000). The above finding also implies that opioid receptors in seizure-regulating forebrain areas may exert some selective stimulatory effects, a proposition that requires further examination but has been already described in hippocampal tissue (Robinson and Deadwyler, 1981; Hampson et al., 2000). Moreover, the putative excitatory effects of opioids may explain the increased susceptibility to forebrainregulated epileptogenesis following chronic opioid treatment (Rocha et al., 1996; Atapour et al., 2000). We further report that a single systemic injection of 1 ng/kg of NTX could induce a significant anticonvulsant effect when combined with morphine at doses as low as 10 ng/kg–100 ␮g/kg. This finding implies a potential novel approach to utilize the potent anticonvulsant effects of opioids in some clinical situations in such low doses that may be devoid of their adverse effects. While this issue should be carefully investigated, some early reports are in favor of the feasibility of treatment with ultra-low doses of opioid antagonists since they block the development of tolerance to morphine analgesia (Crain and Shen, 2000; Powell et al., 2002; Low et al., 2003) and may even decrease the rewarding effects of opioids (Burns et al., 2003; Olmstead, 2003; see however, Powell et al., 2002). It should be also noted that ultra-low doses of NTX did not potentiate the effect of strong anticonvulsant doses of morphine (1 and 3 mg/kg), suggesting a possible ceiling effect for the maximal amount of resistance that may be achieved through modulation of opioid receptors. Further investigation is required to elucidate the exact molecular mechanism of action of ultra low dose NTX, including the possibility of direct interaction with other neurotransmitter systems including GABA. While to our knowledge there is no evidence of direct interaction of ultra low doses of opioid receptor antagonists with GABAergic transmission, opioid agonists like morphine may interact with aspects of GABA transmission including attenuation of nonvesicular GABA release in nucleus accumbens (Schoffelmeer et al., 2001). In contrast to the anticonvulsant effects, the proconvulsant effect of morphine was not significantly affected by ultra-low dose NTX. The proconvulsant effects of high dose opioids has been primarily linked to widespread postreceptor inhibitory effects leading to disinhibition of GABAergic transmission and subsequent increase in excitatory neurotransmitter release (Zieglgänsberger et al., 1979; Sagratella and Massotti, 1982). This is in agreement with the observation that this proconvulsant effect can be blocked by 1 mg/kg of NTX that blocks the main inhibitory

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effects of opioid receptors but is not altered by ultra-low doses of the antagonist that supposedly affect the excitatory signaling. This lack of effect perhaps rules out a potential beneficial effect for ultra-low doses of NTX against clinical seizures induced by very high doses of opioids. In contrast to clonic seizures that are regulated by forebrain centers, generalized tonic– clonic seizures are under brainstem regulation and are considered a model for clinical grand mal seizures (Sarkisian, 2001; Eells et al., 2004). In the present study, the combination of an ultra-low dose of NTX (10 ng/kg) and a non-effective dose of morphine (1 mg/kg) led to a significant increase in the latency and a decrease in the incidence of tonicclonic seizures. The site-specific differential regulatory role of opioid receptors on these two distinct types of seizures may be responsible for the observed difference between the anticonvulsant effects of morphine. However, it should be noted that the i.p. method of PTZ administration has a lower sensitivity compared with the more sensitive i.v. paradigm (Löscher and Schmidt, 1988). Further studies using alternative seizure models that are shown to be modulated by opioids, including the limbic seizures of temporal lobe against which the endogenous opioid dynorphin seems to play a prominent role (Simonato and Romualdi, 1996), are warranted. A further issue of potential interest would be possible use of ultra-low dose NTX as an adjuvant in combination with current anticonvulsant therapies. Two such interactions between opioid receptor modulation and non-opioid pathways have been recently described in antinociceptive paradigms, showing the potentiation of the analgesic effects of a specific serotonin reuptake inhibitor (Singh et al., 2003) and a cannabinoid receptor agonist (Paquette and Olmstead, 2003) by ultra-low dose opioid receptor antagonists. In interpretation of current results, the role of separate subtypes of opioid receptors should be considered. Morphine has affinity for various opioid receptors at high doses but is more selective for ␮ subtype at low doses. It is also known that morphine exerts both its anti- and pro-convulsant effects in PTZ model of seizure through ␮ opioid receptors (Lauretti et al., 1994). NTX is also a non-selective antagonist with more potency for ␮- and ␬- and less for ␦-receptors (Gutstein and Akil, 2001), though at the very low doses used here it is rather selective for ␮-receptors. Nonetheless, interactions between different opioid receptor subtypes play an important role in the modulation of antinociception (Harrison et al., 1998; Vaccarino and Kastin, 2000) as well as in regulation of seizure susceptibility (Frenk, 1983; Frey, 1988; O’Neill et al., 1997; Yajima et al., 2000). Thus, investigation of possible contribution of specific opioid subtypes to the low dose interactions described here would be of interest. In conclusion, present study is the first report of modulation of opioids effects on seizure susceptibility by ultralow dose opioid receptor antagonist. We report that pretreatment with ultra-low dose NTX can be a potent strategy to unmask and potentiate the anticonvulsant effects of low

doses of morphine against clonic seizures without altering its proconvulsant profile.

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(Accepted 15 August 2004)