John E. Pintar

Retention of heroin and morphine–6β–glucuronide analgesia in a new line of mice lacking exon 1 of MOR–1

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR–1 gene. Although morphine–6β–glucuronide (M6G), heroin and 6–acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR–1. Mice homozygous for either MOR–1 mutation were insensitive to morphine. Heroin, 6–acetylmorphine and M6G still elicited analgesia in the exon–1 MOR–1 mutant, which also showed specific M6G binding, whereas M6G and 6–acetylmorphine were inactive in the exon–2 MOR–1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR–1 gene. Although morphine–6β–glucuronide (M6G), heroin and 6–acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR–1. Mice homozygous for either MOR–1 mutation were insensitive to morphine. Heroin, 6–acetylmorphine and M6G still elicited analgesia in the exon–1 MOR–1 mutant, which also showed specific M6G binding, whereas M6G and 6–acetylmorphine were inactive in the exon–2 MOR–1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Potentiation of the D2 mutant motor phenotype in mice lacking dopamine D2 and D3 receptors

Within the D2-class of dopamine receptors, the D2 and D3 subtypes share the highest degree of similarity in their primary structure. However, the extent to which these two receptor subtypes have similar or different functional properties is unclear. The present study used gene targeting to generate mice deficient for D2, D3, and D2/D3 receptors. A comparative analysis of D2 and D3 single mutants and D2/D3 double mutants revealed that D2/D3 double mutants develop motor phenotypes that, although qualitatively similar to those seen in D2 single mutants, are significantly more severe. Furthermore, increased levels of the dopamine metabolites dihydroxyphenyl acetic acid and homovanillic acid are found in the dorsal striatum of D2 single mutants. The levels of these metabolites, however, are significantly higher in mice lacking D2 and D3 receptors. In addition, results of immunoprecipitation experiments revealed that D2 single mutants express higher levels of D3 receptor proteins during later stages of their postnatal development. These results suggest that D3 receptors compensate for some of the lacking D2 receptor functions and that these functional properties of D3 receptors, detected in mice with a D2 mutant genetic background, remain masked when the abundant D2 receptor is expressed.

Neuropathic Pain Activates the Endogenous κ Opioid System in Mouse Spinal Cord and Induces Opioid Receptor Tolerance

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [κ opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the κ agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Differential Activation and Trafficking of μ-Opioid Receptors in Brain Slices

The activation of G protein-coupled receptors results in a cascade of events that include acute signaling, desensitization, and internalization, and it is thought that not all agonists affect each process to the same extent. The early steps in opioid receptor signaling, including desensitization, have been characterized electrophysiologically using brain slice preparations, whereas most previous studies of opioid receptor trafficking have been conducted in heterologous cell models. This study used transgenic mice that express an epitope-tagged (FLAG) μ-opioid receptor (FLAGMOR) targeted to catecholamine neurons by regulatory elements from the tyrosine hydroxylase gene. Brain slices from these mice were used to study tagged MOR receptors in neurons of the locus ceruleus. Activation of the FLAGMOR with [Met5]enkephalin (ME) produced a hyperpolarization that desensitized acutely to the same extent as native MOR in slices from wild-type mice. A series of opioid agonists were then used to study desensitization and receptor trafficking in brain slices, which was monitored with a monoclonal antibody against the FLAG epitope (M1) conjugated to Alexa 594. Three patterns of receptor trafficking and desensitization were observed: 1) ME, etorphine, and methadone resulted in both receptor desensitization and internalization; 2) morphine and oxymorphone caused significant desensitization without evidence for internalization; and 3) oxycodone was ineffective in both processes. These results show that two distinct forms of signaling were differentially engaged depending on the agonist used to activate the receptor, and they support the hypothesis that ligand-specific regulation of opioid receptors occurs in neurons maintained in brain slices from adult animals.

Antinociceptive and Hypothermic Effects of Salvinorin A Are Abolished in a Novel Strain of κ-Opioid Receptor-1 Knockout Mice

Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the κ-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as κ-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 μg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of κ-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional κ-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for κ1- but not κ2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for κ1-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the κ-opioid receptor.

Mu-opioid receptor knockout mice show diminished food-anticipatory activity

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, µ‐opioid receptors are activated. To test this hypothesis schedule induced food‐anticipatory activity in µ‐opioid receptor knockout mice was measured using running wheels. We hypothesized that µ‐knockout mice show little food‐anticipatory activity. In wildtype mice we observed that food‐anticipatory activity increased proportional to reduced food intake levels during daily scheduled food access, and thus reflects the animals physiological need for food. µ‐Knockout mice do not adjust their schedule induced running wheel behaviour prior to and during feeding time in the same way as wildtype mice; rather than showing more running wheel activity before than during feeding, they showed an equal amount of activity before and during feeding. As food‐anticipatory activity is dependent on the mesolimbic dopamine system and µ‐opioid receptors regulate dopaminergic activity, these data suggest a change in the dopamine systems activity in µ‐knockout mice. As we observed that µ‐knockout mice tended to show a stronger locomotor activity response than wildtype mice to the indirect dopamine agonist d‐amphetamine, it appears that the dopaminergic system per se is intact and sensitive to activation. We found no differences in the expression of pro‐opiomelanocortin, a precursor of endogenous endorphin, in the arcuate nucleus between µ‐knockout mice and wildtype mice during restricted feeding, showing that the µ‐opioid receptor does not regulate endogenous endorphin levels. These data overall suggest a role for µ‐opioid receptors in adapting reward related behaviour to the requirements of the environment.

μ-opioid receptor modulation of calcium channel current in periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1

The actions of opioid receptor agonists on the calcium channel currents (IBa) of acutely dissociated periaqueductal grey (PAG) neurons from C57B16/J mice and mutant mice lacking the first exon of the μ‐opioid receptor (MOR‐1) were examined using whole cell patch clamp techniques. These effects were compared with the GABAB‐receptor agonist baclofen. The endogenous opioid agonist methionine‐enkephalin (met‐enkephalin, pEC50 6.8, maximum inhibition 40%), the putative endogenous μ‐opioid agonist endomorphin‐1 (pEC50 6.2, maximum inhibition 35%) and the μ‐opioid selective agonist DAMGO (Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol enkephalin, pEC50 6.9, maximum inhibition 40%) inhibited IBa in 70% of mouse PAG neurons. The inhibition of IBa by each agonist was completely prevented by the μ‐receptor antagonist CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2). The δ‐opioid receptor agonists DPDPE ([D‐Pen2,5]‐enkephalin, 1 μM) and deltorphin II (1 μM), and the κ‐opioid receptor agonist U‐69593 (1–10 μM), did not affect IBa in any cell tested. The GABAB agonist baclofen inhibited IBa in all neurons (pEC50 5.9, maximum inhibition 42%). In neurons from the MOR‐1 deficient mice, the μ‐opioid agonists met‐enkephalin, DAMGO and endomorphin‐1 did not inhibit IBa, whilst baclofen inhibited IBa in a manner indistinguishable from wild type mice. A maximally effective concentration of endomorphin‐1 (30 μM) partially (19%), but significantly (P<0.005), occluded the inhibition of IBa normally elicited by a maximally effective concentration of met‐enkephalin (10 μM). This study indicates that μ‐opioid receptors, but not δ‐ or κ‐opioid receptors, modulate somatic calcium channel currents in mouse PAG neurons. The putative endogenous μ‐agonist, endomorphin‐1, was a partial agonist in mouse PAG neurons.

Tissue-specific expression of the insulin-like growth factor binding protein (IGFBP) mRNAs in mouse and rat development

The insulin-like growth factor binding proteins (IGFBPs) are polypeptides which are thought to modulate the bioactivity of IGF-I and IGF-II, and may also have activities independent of the IGFs. The expression patterns of IGFBPs-1, -3, -4, and -6 in midgestational rodents were analyzed using in situ hybridization to begin to characterize the role of these IGFBPs during development. All IGFBPs are expressed at least as early as rat embryonic day 14 (e14), and each has a unique pattern of expression. IGFBP-1 mRNA is expressed by the liver throughout mid and late gestation. IGFBP-3 mRNA is expressed at high levels in the urogenital tract, several muscle groups, and the nasal epithelia. IGFBP-3 transcripts are also expressed at lower levels by many non-neural tissue types, including the liver, stomach, and heart. IGFBP-4 is abundantly expressed by many tissues in the developing embryo, with the notable exceptions of the spinal cord, specific cartilage groups, and the thymic cortex. IGFBP-6 is expressed in the liver by e14, and also by a previously unrecognized cell population surrounding developing cartilage. Taken together these observations suggest distinct roles in development for each of the IGFBPs.

Nociception increases during opioid infusion in opioid receptor triple knock-out mice

Opioids are extensively used analgesics yet can paradoxically increase pain sensitivity in humans and rodents. This hyperalgesia is extensively conceptualized to be a consequence of opioid receptor activity, perhaps providing an adaptive response to analgesia, and to utilize N-methyl-D-aspartate (NMDA) receptors. These assumptions were tested here in opioid receptor triple knock-out (KO) mice lacking all three genes encoding opioid receptors (mu, delta, and kappa) by comparing their thermal nociceptive responses to the opioids morphine and oxymorphone with those of B6129F(1) controls. Injecting acute opioid bolus doses in controls caused maximal analgesia that was completely abolished in KO mice, confirming the functional consequence of the KO mouse opioid receptor deficiency. Continuous opioid infusion by osmotic pump in control mice also initially caused several consecutive days of analgesia that was shortly thereafter followed by several consecutive days of hyperalgesia. In contrast, continuously infusing KO mice with opioids caused no detectable analgesic response, but only immediate and steady declines in nociceptive thresholds culminating in several days of unremitting hyperalgesia. Finally, injecting the non-competitive NMDA receptor antagonist MK-801 during opioid infusion markedly reversed hyperalgesia in control but not KO mice. These data demonstrate that sustained morphine and oxymorphone delivery causes hyperalgesia independently of prior or concurrent opioid or NMDA receptor activity or opioid analgesia, indicating the contribution of mechanisms outside of current conceptions, and are inconsistent with proposals of hyperalgesia as a causative factor of opioid analgesic tolerance.