No abstract

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Tetrodotoxin was infused into the suprachiasmatic nuclei of unanesthetized and unrestrained rats continuously for 14 days. The internal timekeeping mechanism of the circadian pacemaker in the nuclei continued to oscillate unaffected by this treatment, although the toxin reversibly blocked function of both the input pathway for pacemaker entrainment and an output pathway for expression of the circadian drinking rhythm. Thus, Na+-dependent action potentials appear necessary for entrainment and expression of overt circadian rhythms, but they do not seem necessary for the pacemaker to keep accurate time. The experimental approach presented in this paper is useful because it allows systematic assessment and distinction of the input, pacemaker, and output components of a mammalian circadian timekeeping system in vivo.The suprachiasmatic nuclei (SCN) in the anterior hypothalamus appear to be the site of an endogenous circadian pacemaker in mammals (1). The nuclei receive retinal inputs for entrainment to the environmental light-dark cycle and generate neural outputs for expression of overt, measurable rhythms. Two complementary measures of SCN activity have helped to establish that the nuclei contain a functioning circadian pacemaker. These two properties, in vivo glucose utilization (2, 3) and unit discharge rates (4, 5), exhibit circadian rhythmicity. SCN energy metabolism and electrical activity are both elevated during the day and depressed during the night in nocturnal and diurnal mammals (6, 7).However, the most recent investigations using these two assays have generated some unexpectedly discordant data. On one hand, the rhythm of SCN metabolic activity appears in fetal rats 72 hr before birth (8,9). Such prenatal pacemaker function antedates the postnatal maturation of input and output pathways for photic entrainment and expression of overt circadian rhythms (10). On the other hand, when SCN action potentials are recorded in hypothalamic slices obtained from 7-, 11-, 14-, and 21-day-old rat pups, a circadian rhythm is observed only in those slices from the 14-and 21-day-old animals (11). Although other interpretations are possible, it seems that the circadian rhythm of SCN unit firing rates appears weeks after the rhythm of SCN energy metabolism first begins in utero.To resolve this discrepancy, we propose that the action potentials recorded in the SCN are not a part of the internal timekeeping mechanism of the circadian pacemaker; rather, the electrical impulses function to couple the pacemaker to its input and output pathways. We have tested this idea by chronically infusing tetrodotoxin (TTX) into the SCN of unanesthetized, unrestrained rats. TTX selectively and reversibly blocks voltage-dependent Na+ channels in axons, inhibiting the generation of action potentials without affecting resting membrane potential, K+ currents, Na+ pump mechanism, or local depolarization of postsynaptic membranes (12, 13). Importantly, in vitro recordings in hypothalamic slices demonstrate that SCN action potentials are ...

Mitochondrial Ca2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca 2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A 2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca 2+ from extracellular medium and blockage of mitochondrial Ca 2+ uptake by either a mitochondrial uncoupler or a Ca 2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca 2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca 2+ -dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca 2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.

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 ...

No abstract

Opioid receptors are differentially coupled to ion channels. IA-and 6-opioid receptors are coupled to calciumand/or voltage-dependent potassium channels and K-opioid receptors are coupled to voltage-dependent calcium channels. Using the single-electrode voltage-clamp technique, we investigated the effect of the K-opioid receptor agonist dynorphin A on somatic calcium currents of mouse dorsal root ganglion (DRG) neurons in culture. Three different calcium currents were recorded: a small transient current activated positive to -60 mV; a large, inactivating current activated positive to -50 mV; and a moderate, slowly inactivating current activated positive to -40 mV. The first was less sensitive to cadmium block than the others. These calcium currents were similar to those described in other cells, which have been designated T, N, and L calcium currents, respectively. The opioid peptide dynorphin A reduced calcium current by selectively reducing the large inactivating (N) calcium current. Naloxone, an opioid receptor antagonist, reversed this action of dynorphin A. N calcium current is the predominant calcium current in DRG neurons. If N calcium channels are present in primary afferent terminals, and if they are coupled to K-opioid receptors as in the soma, these results suggest a mechanism by which dynorphin A inhibits calcium influx and neurotransmitter release.Recent studies have established that multiple opioid receptors including ,-, 8-, and K-Opioid receptors are present in the nervous system (1-4) and that they are differentially coupled to ion channels (5-10). ,u-and 3-opioid receptors have been shown to be coupled to potassium channels (5-8, 11, 12 (35)(36)(37). Recently, a third type of calcium current was described in chick DRG neurons (38). The three calcium currents had different activation and inactivation voltage ranges, and the calcium channels had different conductances and kinetics. The currents were differentially affected by the calcium channel blocker cadmium, the agonist dihydropyridine Bay K 8644, and the antagonist dihydropyridines (38)(39)(40). The calcium currents were designated T, N, and L by Nowycky et al. (38).These observations raise the possibility that the K-opioid receptor could be linked to one or more calcium channel types. In the present study we recorded three different somatic calcium currents in mouse DRG neurons and assessed the effect of dynorphin A on these T, N, and L calcium currents. MATERIALS AND METHODSCell Culture. DRG neurons were grown in cell culture as described (41). Spinal cords, with DRG attached, were dissected from 12-to 14-day fetal mice and mechanically dissociated. The resulting suspension was plated onto 35-mm collagen-coated dishes at various densities, usually about one-eighth cord per dish. Initial medium contained 5% horse serum and 5% Nu-Serum (Collaborative Research, Waltham, MA) in minimum essential medium (MEM) (Eagle's; GIBCO). Nerve growth factor (NGF) was added at a final concentration of 10 ng/ml. After 4-5 days in culture, 5'-fluoro...

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.