Morphine and other opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC ␤3-deficient mice, we have established a strong link between PLC and opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC ␤3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED 50 value for morphine in producing antinociception. The reduced ED 50 value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B max and K d values for , , and ␦ opioid receptors between wild-type and PLC ␤3-null mice. We also found that opioid regulation of voltage-sensitive Ca 2؉ channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific agonist [D-Ala 2 ,(Me)Phe 4 , Gly(ol) 5 ]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC ␤3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC ␤3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca 2؉ current reduction. These data demonstrate that PLC ␤3 constitutes a significant pathway involved in negative modulation of opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.Morphine and other opioids are widely used analgesics. Three opioid receptors, , ␦, and , have been cloned and characterized both functionally and by radioligand binding (1-4). These receptors couple to G i and G o proteins and, through ␣ and ␤␥ subunits, regulate a number of signaling pathways. These regulated pathways include inhibition of adenylyl cyclase activity (5, 6), activation of inwardly rectifying K ϩ channels (7,8), and inhibition of voltage-activated Ca 2ϩ channels, predominantly of the N and P͞Q types (9, 10). Recent work with cell lines has demonstrated that opioid receptors also activate phospholipase C (PLC; 11-17). Moreover, several physiological studies have implicated PLC-linked pathways in a diverse range of opioid-modulated events, such as pain regulation (18), response to brain injury (19), and opioid withdrawal (20). However, despite these data suggesting an important role for PLC-mediated pathways in opioid signal transduction, there is little direct evidence supporting a role of PLC in opioid responses.PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce two important second messengers, inositol triphosphate and diacylglycerol (21). The former increases release of Ca 2ϩ from intracellular stores, and the latter activates protein kinase C (PKC). Molecular cloning and biochemical characterizations have revealed four ...

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.

This study examined both the acute and long-lasting changes in seizure susceptibility that occur in response to the repeated induction of generalized seizure activity. Daily flurothyl-induced generalized clonic seizures resulted in a progressive decrease in both the generalized seizure threshold and the latency to the first myoclonic jerk. The threshold reduction was significant as early as the second trial and was maximal by trial 5. However, a minimum of eight seizures was necessary for the maximal reduction to be long-lasting. The present study also examined the effects of the number of seizures and the duration of the stimulation-free interval on the type of generalized seizure expressed. During the induction phase of the experiment, only generalized clonic seizures ("forebrain seizures") were expressed. If, however, the animal was retested after a 1, 2, 3, or 4 week stimulation-free interval, a progressive increase in both the proportion of animals expressing "brainstem seizure" behaviors and the median seizure score was observed. The progression of flurothyl-induced generalized seizure behaviors was significantly altered if (1) a minimum of eight generalized clonic seizures had been expressed, and (2) a minimum of a 2 week stimulation-free interval followed. Fewer generalized clonic seizures failed to reliably produce changes in seizure phenotype, even after extended stimulus-free intervals. These data indicate that specific kindling processes are initiated during the interval of repeated seizure induction and evolve in the absence of continued seizure induction. Furthermore, these mechanisms of epileptogenesis were found to be manifest predominantly as a change in the seizure phenotype expressed and to proceed independent of changes in the generalized seizure threshold.

Diabetic neuropathic pain remains an unmet clinical problem and is poorly relieved by conventional analgesics. N-methyl-D-aspartate (NMDA) receptors play an important role in central sensitization in neuropathic pain. Although NMDA antagonists are highly effective in reducing neuropathic pain, these agents cause severe side effects at therapeutic doses, which limit their clinical uses. Neramexane and memantine are uncompetitive NMDA antagonists with minimal side effects at therapeutic doses. Here we determined the antinociceptive effect of chronic administration of neramexane and compared its effect with that of memantine and gabapentin in a rat model of diabetic neuropathic pain. Mechanical hyperalgesia was measured with a noxious pressure stimulus, and tactile allodynia was assessed with von Frey filaments in diabetic rats induced by streptozotocin. Compared with vehicle-treated rats, treatment with neramexane (12.3, 24.6, and 49.2 mg/kg/day) for 2 weeks via an osmotic minipump produced dose-dependent and sustained effects on mechanical hyperalgesia and allodynia. Administration of memantine (20 mg/kg/day) or gabapentin (50 mg/kg/day) for 2 weeks also produced significant and persistent antinociceptive effects on mechanical hyperalgesia and allodynia. The magnitude of the antinociceptive effect produced by the intermediate and high doses of neramexane was comparable to that of gabapentin and memantine. The plasma level achieved by neramexane at 12.3, 24.6, and 49.2 mg/kg/day was 0.26 ± 0.04, 0.50 ± 0.05, and 1.21 ± 0.16 μM, respectively. These data suggest that neramexane at therapeutically relevant doses attenuates diabetic neuropathic pain. Our study provides valuable information about the therapeutic potential of chronic administration of neramexane and memantine for painful diabetic neuropathy.

Summary: Purpose:We have developed and characterized a novel model of epileptogenesis based on the convulsive actions of flurothyl in mice. The hallmark feature of this model is a reliable change in the type of seizure expressed in response to flurothyl from generalized clonic to generalized tonic seizures. The purpose of our study was to evaluate the effects of chronic administration of valproate (VPA), phenytoin (PHT), and MK-801 on the change in seizure phenotype observed in our model system.Methods: Male C57BW6J mice received flurothyl seizures on 8 consecutive days. Two hours after the last generalized seizure, chronic drug or vehicle was administered twice daily at 12-h intervals for 28 days. The drugs evaluated were VPA (250 mgkg), PHT (30 mgkg), and MK-801 (0.5 mgkg). After a 7-day drug washout period, mice were retested with flurothyl.Results: Among uninjected or vehicle-injected control mice, there was a significant increase in the proportion of animals expressing tonic seizures after the 28-day stimulation-free interval. Chronic administration of VPA or MK-801, but not PHT, blocked the characteristic change in seizure type from clonic to tonic. Conclusions:The change in seizure phenotype observed after exposure to our paradigm indicates a fundamental reorganization in the propagation of flurothyl-initiated seizures. As in electrical kindling, VPA and MK-801 are effective at blocking or retarding the reorganization, whereas PHT is not. The concordance in pharmacologic profiles between kindling and our model suggests that the processes underlying changes in seizure susceptibility in these two models share mechanisms in common. Key Words: Flurothyl-Generalized seizuresSeizure propagation-Seizure threshold-Epileptogenesis-C57BL/6J mice.A significant body of research indicates that two, largely independent networks in the rodent brain mediate the expression of experimentally induced seizures (1-5). A low-threshold forebrain system mediates expression of generalized clonic seizures, and a highthreshold brainstem system mediates expression of generalized tonic seizures. Propagation of seizure activity in the forebrain system results in the expression of seizures characterized by clonus of the face and forelimbs. By contrast, propagation of seizure activity in the brainstem system results in seizures characterized by tonus in the forelimbs and hindlimbs. Hypothetically, the process of epileptogenesis would involve alterations in the networks responsible for seizure expression. These could involve alterations in the threshold for seizure initiation in either system or alterations in the propagation of seizure activity in either network or between the forebrain or brainstem seizure networks.The process or processes underlying the development of epilepsy are largely unknown, and relatively few animal models are available for investigation of epileptogenic processes. We recently developed an experimental model system in mice that allows detection of a longlasting change in seizure susceptibility and which therefo...