362 Experiments with adenosine receptor agonists and antago- nists strongly indicate that the turning reaction is caused by activation of adenosine receptors. Adenosine receptors belong to the large family of seven transmembrane G protein-coupled receptors [8], which contain members known to be involved in growth-cone turning [9, 10]. The finding that micro-gradients of ADO induce growth- cone turning is unexpected because of negative results in comparable experiments [6, 44]. This apparent discrepancy with our results may be explained by experimental differ- ences. First of all, the data published so far are derived from spinal neurons of Xenopus, whereas we investigated sensory neurons from chicken. Another difference is the concentra- tion of the agonists used in the studies mentioned above. Fu and colleagues [6] used a pipette solution containing a 30- fold higher concentration of ADO than in the present study, and Stein et al. [44] performed the experiments with a 100- fold higher concentration of NECA compared to the concentration in this work. Even though it is hard to compare micro-gradients used by different labs just on the basis of published data, we estimate that the agonist concentrations used in the former studies were significantly higher than those used in this work, not only in the pipette solution but also at the site of the growth cones. Our finding that micro-gradients produced by micro-pipettes with an increased tip diameter did not attract growth cones supports the interpretation that high concentrations counteract the attractive effect of ADO. It is known from the work of Goodhill [17] that gradient detection is disturbed if the agonist concentration is high compared to the dissociation constant for the receptor–ligand complex. In this case almost all receptors are bound, hence yielding little difference in binding across the extent of the growth cone. Another reason for the ineffectiveness of high ADO concentrations might be that in case of adenosine receptors, one cell can express several receptor subtypes with different binding constants and diverse physiological effects. Therefore, at high ADO concentrations the summed response of different adenosine receptor subtypes may not favour growth-cone turning. Homologues to all four known human ADO receptors have been found in the chicken genome [45–48]. It has been shown that A1 and A2A receptors are present at sensory neurons of rodents [49, 50]. We are not aware of published data showing the presence of ADO receptors on chicken sensory neurons, but all experiments with agonists and antagonists shown here indicate that ADO receptors are present on growth cones of chicken sensory neurons and are responsible for the observed growth-cone turning response. The affinities of the different adenosine receptor subtypes range from an adenosine concentration of ~70 nM for the A1 receptor up to ~6,500 nM for the A3 receptor subtype [38, 51]. Here we show that micro-gradients effective in growth-cone turning have an adenosine con- Purinergic Signalling (2008) 4:357–364 centration between 60 and 100 nM. Consequently, the best candidates for mediating the adenosine response shown here are the high affinity receptors, namely the A1 (~70 nM) and the A2A (~150 nM) receptor. Experiments with the subtype-specific adenosine receptor agonist CGS 21680 [40] indicate a function of the A2A receptor in adenosine-induced growth-cone turning, whereas no re- sponse was detectable with the usage of the A1 receptor specific agonist R-(-)-PIA [37]. The latter result argues against a major contribution of the A1 receptor in the observed turning response. However, remarkable species differences have been observed for adenosine receptor agonists [37], and chicken adenosine receptors are pharma- cologically not well characterised. Therefore future experi- ments with ADO receptor subtype-specific ligands have to clarify whether A2A receptor activation is necessary or sufficient to induce growth-cone turning in sensory neu- rons. In particular antagonists (e.g. SCH 58261 [52]) seem interesting due to the fact that they remove a single player from the set of different adenosine receptor subtypes that could be present on the growth cone. ADO receptor signalling is classically thought to occur through changing the intracellular cAMP level [53]. Indeed the presented results show that the cAMP antagonist Rp- cAMPS blocks growth-cone turning induced by ADO or the A2A specific agonist CGS 21680. However no clear picture has emerged by testing a possible involvement of PKA, an important downstream effector of cAMP. The PKA-specific inhibitor KT 5720 [43] has different effects, depending on the used chemoattractant. KT 5720 blocks the attractant effect of ADO, but not of CGS 21680. One explanation for this phenomenon could be that ADO and CGS 21680 activate different subsets of the four known ADO receptors. As ADO receptors couple to an intricate network of signalling pathways [54], the activation of different receptor subsets could be the reason for the observed difference in sensitivity to KT 5720. Off-target effects of CGS 21680 to proteins other than ADO receptors could be an alternative explanation and show the need for future thorough pharmaceutical characterisation of the signal transduction chain. Besides PKA as a target of cAMP, the importance of ‘exchange protein activated by cyclic AMP’ (EPAC) has also become generally known [55]. It has been shown that activation of EPAC can promote neurite extension in PC12 cells and that EPAC acts in synergy with PKA during this process [56]. On this basis one might speculate that EPAC could also be involved in ADO-induced growth-cone turning. Our finding that cAMP participates—at least to some extent—in the signal transduction chain of ADO- induced growth-cone turning is not unexpected, as exam- ples of cAMP-dependent growth-cone turning have been published [10, 14]. On the other hand, a contribution of