Benjamin Kest

N-Methyl-D-Aspartate (NMDA) Receptors, Mu and Kappa Opioid Tolerance, and Perspectives on New Analgesic Drug Development

This laboratory perspective reviews the pharmagologic approaches that have been used in preclinical animal models to demonstrate the ability of competitive (LY274614) and noncompetitive (MK801 and dextromethorphan) N-methyl-D-aspartate (NMDA) receptor antagonists to attenuate or reverse the development of morphine tolerance. We provide additional data to support previous observations that these NMDA antagonists modulate morphine (mu) opioid tolerance but do not affect U50488H (kappa1) opioid tolerance. A strategy, which utilizes efficacy as an NMDA receptor antagonist and clinical safety, provides the basis for a discussion of the clinical potential of dextromethorphan, ketamine, and felbamate as modulators of opioid tolerance in pain patients or opioid addicts. The potential use of NMDA receptor antagonists and nitric oxide synthase (NOS) inhibitors in neuropathic pain is also discussed.

An antisense oligodeoxynucleotide to the delta opioid receptor (DOR-1) inhibits morphine tolerance and acute dependence in mice

Pharmacological data from several laboratories support a modulatory role for the delta opioid receptor in morphine analgesia, tolerance, and physical dependence. We examined the role of the delta opioid receptor in these processes using an in vivo antisense strategy in mice. Intracerebroventricular administration of a 20mer antisense or a mismatch control oligodeoxynucleotide (ODN) targeting the mRNA of the cloned delta opioid receptor (DOR-1) for 3 days did not affect baseline nociceptive thresholds or morphine analgesia compared to untreated or saline-treated mice. However, dose-response studies indicate that the induction of morphine tolerance following 3 days of chronic morphine administration was blocked in antisense but not mismatch ODN or saline-treated mice. Antisense ODN treatment also blocked the development of acute morphine dependence, whereas similar protection was not afforded to mice treated with saline or mismatch ODN. This study demonstrates the relevance of the cloned DOR-1 in morphine tolerance and dependence and provides new evidence for a modulatory role of the delta opioid receptor using this novel approach.

Neonatal testosterone exposure influences neurochemistry of non-opioid swim stress-induced analgesia in adult mice

&NA; The effects of neonatal hormone manipulations on swim stress‐induced analgesia (SSIA) magnitude and neurochemical quality were examined in Swiss‐Webster mice of both sexes. Previous research has indicated that non‐opioid SSIA mechanisms in adult Swiss‐Webster mice are sexually dimorphic. Male mice exhibit non‐opioid SSIA following a 3‐min swim in cold (15°C) water that is antagonized by the non‐competitive NMDA antagonist MK‐801 (dizocilpine; 0.075 mg/kg), whereas female mice do not display NMDA‐mediated analgesia in the presence of estrogen. Since male and female mice show equipotent magnitudes of SSIA, it was concluded that female mice display a neurochemically distinct, estrogen‐dependent SSIA mechanism specific to their gender. In the present study, female mice exposed to testosterone during the neonatal period display NMDA‐mediated analgesia even in the presence of estrogen in adulthood. Thus, expression of the female‐specific, estrogen‐dependent SSIA mechanism previously described may be dependent on the absence of testosterone during early ontogeny.

The effects of LY293558, an AMPA receptor antagonist, on acute and chronic morphine dependence

In rodents, noncompetitive and competitive NMDA receptor antagonists have been shown to attenuate and, in some cases, reverse tolerance to the analgesic effects of morphine. However, the ability of these same excitatory amino acid (EAA) receptor antagonists to modulate morphine dependence is controversial, and very little is known about the role of AMPA receptors in morphine dependence. LY293558, a novel, systemically active, competitive AMPA receptor antagonist and the NMDA receptor antagonists, MK-801 and/or LY235959, were evaluated in tolerant or dependent CD-1 mice. In mice rendered tolerant by morphine injection or pellet implantation, continuous s.c. infusion of LY293558 (60 mg/kg per 24 h) or MK-801 (1 mg/kg per 24 h) attenuated the development of tolerance. Neither LY293558 nor MK-801 produced analgesia or altered the ED50 value of morphine. Continuous s.c. infusion of LY293558 (60 mg/kg per 24 h), MK-801 (1 mg/kg per 24 h) or LY235959 (12 mg/kg per 24 h) attenuated the development of acute (3 h) morphine dependence (i.e., decreased naloxone-precipitated withdrawal jumping). In contrast, continuous s.c. infusion of LY293558 (60 mg/kg per 24 h) or LY235959 (12 mg/kg per 24 h) did not significantly attenuate the development of chronic dependence produced by morphine pellet implantation. These data indicate that the development of morphine tolerance is more sensitive to modulation by EAA receptor antagonists than is the development of morphine dependence as assessed by naloxone-precipitated withdrawal jumping.

Assessment of delta opioid antinociception and receptor mRNA levels in mouse after chronic naltrexone treatment

The antinociceptive potency of the delta opioid receptor (DOR) agonist [D-Ala2]Deltorphin II and the levels of DOR mRNA were measured in mice chronically treated with naltrexone. ED50 determinations for [D-Ala2]Deltorphin II, using the tail-flick test with mice that had been treated with naltrexone for 7 days followed by a 24 h naltrexone free period (study day 8), revealed a 7.7-fold increase in antinociceptive potency, indicating functional supersensitivity. Utilization of a micro-dissection technique followed by quantitative solution hybridization measurements of RNA extracts from mouse CNS revealed levels of DOR mRNA ranging from 2.8 pg/microgram RNA in the caudate-putamen to 0.3 pg/microgram RNA in cerebellum. However, despite the functional increase in DOR sensitivity, the DOR mRNA levels in selected brain areas and spinal cord of naltrexone-treated and control mice did not differ. Assessment of DOR mRNA levels in whole brain and selected CNS regions after shorter treatment intervals (1, 6 and 12 h and 2 and 7 days) in naltrexone-treated and control mice revealed a similar pattern of results. Northern blot analysis of mouse whole brain RNA extracts after 7 days of naltrexone treatment did not show any alteration in the size of the DOR transcript. These data demonstrate that DOR mRNA levels are not altered during and after chronic naltrexone treatment and therefore are not associated with opioid-induced DOR up-regulation or DOR functional supersensitivity.

Quantitative distribution of the delta opioid receptor mrna in the mouse and rat CNS

We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to quantitate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 +/- 0.1 (SEM) pg/micrograms RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/micrograms RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/micrograms RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/micrograms RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 +/- 0.1 pg equivalents/micrograms RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/micrograms RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/micrograms RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.

Chronic naltrexone differentially affects supraspinal δ-opioid receptor-mediated antinociception

Abstract The effects of chronic treatment with naltrexone, an opioid receptor antagonist, on δ1- and δ2-opioid receptor agonist-induced antinociception and ligand binding were investigated in mice. Antinociception by intracerebroventricular (i.c.v.) [ d -Pen2,5]enkephalin (DPDPE) and [ d -Ala2]deltorphin II, agonists selective for δ1- and δ2-opioid receptors, respectively, was blocked following subcutaneous (s.c.) implantation of a naltrexone pellet (7.5 mg) for 7 days. Removal of the naltrexone pellet was followed 24 h later by a decrease of 7.5-fold in the ED50 value of [ d -Ala2]deltorphin II, but not that of DPDPE. In a whole brain homogenate the binding of [ 3 H ][ d -Ala2]deltorphin II was increased twice as much as that of [ 3 H ]DPDPE. Chronic naltrexone treatment also produced an 8.6-fold decrease in the ED50 value of i.c.v. administered morphine. The increase in morphine potency was reversed to a control (placebo-treated mice) value by the selective δ2-opioid receptor antagonist, naltriben (25 pmol, i.c.v.). Thus, chronic naltrexone selectively increases δ2-opioid receptor-mediated antinociception, supporting the existence of δ opioid receptor subtypes with distinct adaptive characteristics. The data also indicate that δ2-opioid receptors are critically involved in the expression of morphine supersensitivity.

Effect of supraspinal antisense oligodeoxynucleotide treatment on δ-opioid receptor mRNA levels in mice

Studies in vivo demonstrate that antisense oligodeoxynucleotide (ODN) treatment specifically reduces the functions mediated by numerous central nervous system (CNS) receptors, including opioid receptors. However, the effects of antisense ODN on the opioid receptor mRNA target, itself are rarely examined. In the present study, the effect of supraspinal antisense ODN administration on delta-opioid receptor (DOR) mRNA levels in selected CNS regions, was investigated in mice. ODN targeting a 20-nucleotide sequence of the DOR mRNA transcript was administered by intracerebroventricular (i.c.v.) injection twice daily for 3 days. First, to confirm that antisense ODN treatment decreases DOR function in this system, antinociception produced by DOR-selective agonist [D-Ala2]deltorphin II was assessed on day 4. A 2-fold reduction in [D-Ala2]deltorphin II potency was revealed in antisense ODN-treated mice compared to mice receiving control treatments. DOR mRNA levels in selected CNS regions which either mediate antinociception; medial thalamus (MThal), periaqueductal gray (PAG), frontal cortex (FCtx) and spinal cord (SpC) or exhibit relatively high levels of DOR mRNA; nucleus accumbens (Acb) and caudate-putamen (CPu) were then quantitated by solution hybridization. Levels of DOR mRNA in antisense ODN-treated mice were not different from levels in mice treated with saline vehicle, which ranged from 0.07 pg/microg total RNA in MThal and PAG to 0.26 pg/microg total RNA in CPu. These results are both consistent with previous reports that antisense oligodeoxynucleotide (ODN) treatment down-regulates DOR protein in vivo and indicate that this down-regulation is not associated with altered DOR mRNA levels.

Blockade of morphine supersensitivity by an antisense oligodeoxynucleotide targeting the delta opioid receptor (DOR-1).

An antisense oligodeoxynucleotide (ODN) targeting 20 bases of the coding sequence of the cloned delta opioid receptor (DOR-1), a mismatched ODN (different from the antisense ODN at 4 bases) or saline was administered to 3 groups of CD-1 mice implanted with naltrexone pellets (7.5 mg) for 7 days. Morphine supersensitivity (i.e., increased potency as defined by decreased morphine ED50 values) was observed 24 h after pellet removal (day 8) in mice treated with saline or mismatch ODN, but not in antisense ODN treated mice. Antisense ODN alone had no effect on basal nociceptive thresholds or morphine analgesia but reduced the analgesic potency of the delta, opioid agonist [D-Ala2]deltorphin II. These data suggest that the delta2 opioid receptor system participates in the adaptive changes contributing to increased morphine potency following chronic naltrexone treatment.