Nitric oxide regulates growth cone filopodial dynamics via ryanodine receptor-mediated calcium release Kristy Welshhans and Vincent Rehder Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302, USA Keywords: cADPR, calcium influx, Helisoma trivolvis, PKG, sGC Abstract Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca 2+ ] i ). Moreover, these NO- induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca 2+ ] i that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO- induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process. Introduction Nitric oxide (NO) is a gaseous messenger which has multiple functions during development, including regulation of proliferation, differentiation, pathfinding and synaptic pruning (Ernst et al., 1999; Cogen & Cohen-Cory, 2000; Contestabile & Ciani, 2004; Matarre- dona et al., 2005; Bicker, 2006; Villalobo, 2006). The signalling pathways through which NO brings about these various effects are only partially understood. We have employed a cell culture approach, using identified neurons from the pond snail Helisoma trivolvis, to study how NO affects neuronal motility, with a particular focus on the effects of NO on growth cone dynamics. Growth cones are the motile tips of advancing neuronal processes, such as axons and dendrites, and play a crucial role in neuronal pathfinding and synaptogenesis. NO affects several aspects of neurite outgrowth and targeting (Cheung et al., 2000; Ernst et al., 2000; Haase & Bicker, 2003; Zhang et al., 2005; Bicker, 2006), making the growth cone a highly sensitive assay system for investigating NO signalling. Previous studies from this lab have demonstrated that the application of NO donors, such as SIN-1 and 3-(2-Hydroxy-1-methyl-2- nitrosohydrazino)-N-methyl-1-propanamine (NOC-7), to a particular type of neuron (B5) grown in cell culture resulted in a transient increase in filopodial length, a decrease in filopodial number and a slow-down in neurite outgrowth (Van Wagenen & Rehder, 1999; Trimm & Rehder, 2004; Welshhans & Rehder, 2005). These effects are elicited by a NO-induced transient increase in the intracellular calcium concentration ([Ca 2+ ] i ) and amount to what we have termed growth cone ‘slow down and search behaviour.’ Using a puffer- pipette approach we subsequently demonstrated that the effects on growth cone morphology and calcium were mediated at and limited to the stimulated growth cone (Welshhans & Rehder, 2005). Using pharmacological tools, we determined that NO causes these changes in growth cone morphology via an intracellular cascade involving soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP), protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). The goal of the present study was to further extend the investigation of the NO signalling pathway downstream of cADPR in order to link the effect of NO to the increase in [Ca 2+ ] i , which is a necessary step in the NO-induced changes in growth cone motility. We here report that NO acted via cADPR and ryanodine receptor (RyR)-mediated intracellular calcium release to cause the transient increase in [Ca 2+ ] i and changes in growth cone morphology. The calcium release from intracellular stores then resulted in calcium influx across the plasma membrane, which contributed to the transient intracellular calcium increase seen after stimulation of growth cones with NO. Taken together, this study demonstrates that NO can directly affect growth cone motility of sGC-containing neurons through a transient elevation of [Ca 2+ ] i from ryanodine (Ry)-sensitive stores, making NO an effective signalling molecule at the growth cone level. Thus, growth cones from NO-sensitive neurons could be affected during neuronal pathfinding or synaptogenesis by individual cells or tissues that release NO in the vicinity of a growth cone’s migratory path. Correspondence: Dr Vincent Rehder, as above. E-mail: vrehder@gsu.edu Received 11 May 2007, revised 2 July 2007, accepted 16 July 2007 European Journal of Neuroscience, Vol. 26, pp. 1537–1547, 2007 doi:10.1111/j.1460-9568.2007.05768.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd