Purinergic Signalling (2008) 4:357–364 cAMP-independent mechanisms cannot be excluded on the basis of the current data. The present results show that concentration gradients of ADO can induce changes in the growth direction of neurons, raising the question of sources and sinks of extracellular ADO. Extracellular ADO is either released from cells via transporters or generated from extracellular adenine nucleo- tides by the action of ectonucleotidases [19–21]. Not only ADO sources, but also the mechanisms for removal of ADO from the extracellular space are diverse and widely distributed [20, 38]. Therefore, it can be assumed that ADO concentration gradients that evolve between ADO sources and sinks are widespread. Direct measurements of such gradients are rare, but in a report of Chuna [18], evidence has been provided for an ADO concentration gradient that surrounds electrically stimulated hippocampal neurons. At the moment only in vitro data on ADO-induced growth-cone turning are available, so one can only speculate on the physiological role and significance of the observed effect. However, as ADO is known to have significant effects in wound healing [57], it might also support the reinervation of tissue with sensory neurons after lesions. Acknowledgements We thank G. Wanner and E. Facher for scanning electron microscopic analysis of the pipettes, E. Bartl for her work during the initial phase of the project, TILL Photonics (now part of Agilent Technologies) for assistance with TIRF microscopy, and Dave Vogel for his valuable comments on the text. This work was funded by grants from the Bundesministerium für Bildung und Forschung within the ‘Biophotonik’ programme (FKZ 13N8780 and FKZ 13N9267). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Goldberg JL (2003) How does an axon grow? Genes Dev 17:941– 958. doi:10.1101/gad.1062303 2. Höpker VH, Saffrey MJ, Burnstock G (1996) Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth- promoting effect of myenteric plexus explants. Int J Dev Neurosci 14:439–451 3. Schäfer KH, Saffrey MJ, Burnstock G (1995) Trophic actions of 2-chloroadenosine and bFGF on cultured myenteric neurones. Neuroreport 6:937–941 4. Rathbone MP, Middlemiss PJ, Gysbers JW et al (1999) Trophic effects of purines in neurons and glial cells. Prog Neurobiol 59:663–690. doi:10.1016/S0301–0082(99)00017–9 5. Zimmermann H (2006) Nucleotide signaling in nervous system development. Pflugers Arch 452:573–588. doi:10.1007/s00424– 006–0067–4 6. Fu WM, Tang YB, Lee KF (1997) Turning of nerve growth cones induced by the activation of protein kinase C. Neuroreport 8:2005–2009 363 7. Chen Y, Corriden R, Inoue Y et al (2006) ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science 314:1792–1795. doi:10.1126/science.1132559 8. Horn F, Vriend G, Cohen FE (2001) Collecting and harvesting biological data: the GPCRDB and NucleaRDB information systems. Nucleic Acids Res 29:346–349. doi:10.1093/nar/29.1.346 9. Xiang Y, Li Y, Zhang Z et al (2002) Nerve growth cone guidance mediated by G protein-coupled receptors. Nat Neurosci 5:843– 848. doi:10.1038/nn899 10. Guirland C, Buck KB, Gibney JA et al (2003) Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide. J Neurosci 23:2274–2283 11. Fredholm BB, Ijzerman AP, Jacobson KA et al (2001) Interna- tional Union of Pharmacology. XXV. Nomenclature and classifi- cation of adenosine receptors. Pharmacol Rev 53:527–552 12. Cunha RA (2001) Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. Neurochem Int 38:107– 125. doi:10.1016/S0197–0186(00)00034–6 13. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492 14. Munck S, Bedner P, Bottaro T et al (2004) Spatiotemporal properties of cytoplasmic cyclic AMP gradients can alter the turning behaviour of neuronal growth cones. Eur J Neurosci 19:791–797. doi:10.1111/j.0953–816X.2004.03118.x 15. Lohof AM, Quillan M, Dan Y et al (1992) Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J Neurosci 12:1253–1261 16. Song HJ, Poo MM (2001) The cell biology of neuronal navigation. Nat Cell Biol 3:E81–E88. doi:10.1038/35060164 17. Goodhill GJ (1998) Mathematical guidance for axons. Trends Neurosci 21:226–231. doi:10.1016/S0166–2236(97)01203–4 18. Cunha RA (1997) Release of ATP and adenosine and formation of extracellular adenosine in the hippocampus. In: Okada Y (ed) The role of adenosine in the nervous system. Elsevier, Amsterdam, pp 135–142 19. Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362:299–309. doi:10.1007/s002100000309 20. King AE, Ackley MA, Cass CE et al (2006) Nucleoside transporters: from scavengers to novel therapeutic targets. Trends Pharmacol Sci 27:416–425. doi:10.1016/j.tips.2006.06.004 21. Schwiebert EM, Zsembery A, Geibel JP (2003) Cellular mecha- nisms and physiology of nucleotide and nucleoside release from cells: current knowledge, novel assays to detect purinergic agonists, and future directions. In: Schwiebert EM (ed) Extracel- lular nucleotides and nucleosides: release, receptors, and physio- logical and pathophysiological effects. Academic Press, Amsterdam, pp 31–58 22. Gallo G, Lefcort FB, Letourneau PC (1997) The trkA receptor mediates growth cone turning toward a localised source of nerve growth factor. J Neurosci 17:5445–5454 23. Adams DN, Kao EY, Hypolite CL et al (2005) Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide. J Neurobiol 62:134–147. doi:10.1002/neu.20075 24. Cao X, Shoichet MS (2003) Investigating the synergistic effect of combined neurotrophic factor concentration gradients to guide axonal growth. Neuroscience 122:381–389. doi:10.1016/ j.neuroscience.2003.08.018 25. Dontchev VD, Letourneau PC (2003) Growth cones integrate signaling from multiple guidance cues. J Histochem Cytochem 51:435–444 26. Nishi R (1996) Autonomic and sensory neuron cultures. In: Bronner-Fraser M (ed) Methods in cell biology, methods in avian embryology, vol 51. Academic Press, San Diego, pp 249–263