Potentiation of transmitter release by MU-delta- and kappa-opioids involves modulation of N-type voltage-dependent calcium channels

S28 POTENTIATION OF TRANSMITTER RELEASE BY MU-, DELTA- AND KAPPA-OPIOIDS INVOLVES MODULATION OF N-TYPE VOLTAGE-DEPENDENT CALCIUM CHANNELS O. Keren, M. Gafni, Y. Sarne Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel The regulation of neurotransmitter release by opioids was examined in two human neuroblastoma cell-lines: SK-N-SH, which expresses mu and delta opioid receptors, and NMB, which expresses delta, kappa and mu opioid receptors. Opioids induce dual (inhibitory and excitatory) regulation of depolarization-evoked 3H-dopamine release in SK-N-SH cells through either mu or delta receptors. The potentiation of dopamine release by opioid agonists is mediated by N-type voltage dependent calcium channels, and does not involve Gi/Go proteins. Removal of the excitatory opioid effect by blockade with omega-conotoxin, an Nchannel antagonist, reveals the ‘classic’ inhibitory effect of opioids on release. Activation of kappa-opioid receptors in NMB cells potentiates both basal and depolarization-evoked 3H-dopamine release. The potentiation of dopamine release by the kappa agonist U50,488 is abolished by the N-channel antagonist omega-conotoxin. Removal of the excitatory effect of U50,488 by omega-conotoxin reveals inhibitory regulation of release, similar to our findings in SK-N-SH cells. These findings suggest (1) that potentiation of transmitter release through each of the opioid receptor subtypes (mu, delta, kappa) is mediated by N-type voltage dependent calcium channels, and (2) that both modulatory effects of opioids (excitatory and inhibitory) are exerted in parallel, as the removal of one component (excitatory) reveals the existence of the opposite (inhibitory) effect. NEURONAL NOS ASSOCIATION TO CAVEOLAE-LIKE DOMAINS IN ADULT MICE DEPENDS ON THE EXPRESSION OF THE CELLULAR PRION PROTEIN G.I. Keshet, H. Ovadia and R. Gabizon Dept. of Neurology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel PrPSc, the only identified component of the scrapie prion, is a conformational isoform of PrPc (cellular prion protein). The physiological c role of PrP , a glycoprotein anchored to neuronal cell surface, is still unknown. PrP ablated mice (PrP0/0) develop and reproduce normally; however, they show some neurological abnormalities. We have shown recently that the activity of neuronal nitric oxide synthase (nNOS) was decreased in scrapie infected brains. NO, a gaseous free radical neurotransmitter, is involved in synaptic transmission in the CNS. The aim of this study was to investigate whether nNOS reduced activity in scrapie c infected brains can be related to insufficient function of PrP . Here we report that nNOS presents with impaired activity in adult PrP0/0 mice. We found that in wild-type (WT) mice of different ages nNOS partitions to caveolae-like domains (CLDs), in which PrP resides. However, in scrapie infected mice and in adult PrP0/0 mice nNOS did not partition into caveolae-like domains (CLDs) in the brain. We conclude that c PrP may be involved in the process of targeting nNOS to its proper c subcellular location, and that the function of PrP is probably impaired in scrapie infected brains. CONFOCAL MICROSCOPE IMAGING OF MICROTUBULE REMODELING BY CALPAIN ASSOCIATED TO RETRIEVED VESICLES DURING GROWTH CONE FORMATION AFTER AXOTOMY A. Khoutorsky, D. Gitler, and M. E. Spira The Hebrew University of Jerusalem, Jerusalem Israel and the Interuniversity Institute for Marine Sciences, Eilat, Israel The transformation of a differentiated adult axon into a motile growth cone after axotomy involves massive structural modifications. Using primary cultures of Aplysia neurons, we have shown that the

dedifferentiation of the axon is triggered by a local elevation of the calcium concentration. Confocal microscope imaging of calpain activity and of microtubules post axotomy revealed that: Calpain activity accumulates and dissipates at the growth cone’s center with the same time course as that of endocytozed vesicles. Calpain activity is distributed in the growth cone in a patchy granulated manner that overlaps with the distribution of the retrieved vesicles. These observation suggests that the proteolytic activity is associated with the membranes of the retrieved vesicles rather than being in a soluble form in the cytosol. The association of calpain with the densely packed retrieved membrane forms a ‘hot spot’ of proteolytic activity. Imaging of the distribution of microtubules revealed that axotomy induces rapid depolymerization of the microtubules at the tip of the transected axon. The microtubules then reassemble in the axon’s cortex, but not at the ‘hot spots’ of calpain activity. This spatial relations suggest that the proteolytic activity associated with the retrieved vesicle pool forms a microtubule free space in which the vesicles accumulate. As vesicles are shipped to the periphery, the dimensions of the microtubule-free space is reduced and the cytoskeleton reassembles. These findings provide a tentative explanation for the dynamic relations between the cytoskeletal organization and the sites of vesicle accumulation in growth cones and synapses. DISLODGMENT AND ACCELERATED DEGRADATION OF RAS Y. Kloog1, M. Gana-Weisz1, H. Niv1, G. Elad1, D. Marciano2 and R. Haklai1 1 Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel; 2 Israel Institute for Biological Research, Ness Ziona, Israel Ras proteins are essential components of receptor-mediated signaling cascades that regulate cell growth and differentiation in a variety of types of mammalian cells. This explains why abnormal Ras functions are associated with diverse human diseases including cancer and disorders of the immune and of the nervous system. In attempts to block the undesired actions of activated Ras proteins we have developed new synthetic compounds that interfere with membrane anchorage of Ras, which is required for Ras-dependent signaling. Among these compounds S-trans,trans-farnesylthiosalicylic acid (FTS) and its geranylgeranyl, but not its geranyl analogue, proved to be potent Ras antagonists. FTS inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf transformed cells, suggesting that it interferes specifically with Ras functions. We now demonstrate that FTS dislodges Ras from the membranes of Ha-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras, The dislodged Ras that is transiently present in the cytosol is degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. Thus, FTS appears to act as a true Ras antagonist. Indeed, FTS interferes with the activation of ERK, and with signaling to actin-cytoskeleton. FTS also inhibits ERK activity in fibroblasts stimulated by serum, EGF or thrombin and in PC12 cells stimulated by NGF and carbamylcholine. The results suggest that FTS competes in a rather specific manner with Ras for docking domains in the cell membrane. In support of these conclusions, preliminary results now show specific actions of FTS on the organization of Ras in the plasma membrane. ELECTROTONIC COUPLING IN THE INFERIOR OLIVARY NUCLEUS AND ITS FUNCTIONAL ORGANIZATION A. Kogan and Y. Yarom Dept. of Neurobiology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel The membrane potential of mature inferior olivary (IO) neurons oscillates spontaneously at a frequency of up to 10 Hz. This subthreshold oscillatory activity is postulated to play a major role in the organization of rhythmic behavior of the cerebellar cortex. Using brain slice preparations from 14–30 day old rats, simultaneous double whole-cell patch