Purinergic Signalling (2008) 4:357–364 361 Fig. 3 Effects of different adenosine receptor agonists and antagonists on growth-cone turning. All experiments were performed with 1-μm pipettes filled with the respective agonist or 10 μl/ml DMSO for the control. Both ADO (100 μM) and the unspecific adenosine receptor agonist NECA (10 μM) induced a significant positive turning response. In the presence of the unspecific adenosine receptor antagonist CGS 15943, no ADO-induced turning response was detectable. CGS 15943 was added to the culture medium and to the pipette solution in a concentration of 10 μM. The A1 receptor specific agonist R-(-)-PIA (10 μM) caused no significant turning response. In contrast to this, a positive turning response was induced by the specific A2A receptor agonist CGS 21680 (10 μM). Displayed are the average turning angles, error bars indicate standard error of the mean (SEM), and n the sample size. Significant differences from the control group are marked (* P<0.05; ** P<0.01; Mann-Whitney U test) turning (data not shown). As the absence of a turning response can always be caused by inappropriate gradient shape, we tested whether a concentration increase to 10 μM in the stimulating pipette has any effect (Fig. 3). However, this condition was as ineffective as a decreased start distance (35 μm instead of 50 μm) between growth cone and a micro-pipette containing 2 μM R-(-)-PIA (data not shown). In summary, we have no indication that a growth- cone turning response can be activated by R-(-)-PIA. In contrast to this, a pipette filled with 10 μM of the A2A receptor specific agonist CGS 21680 [40] induced a significant attractant response. These results provide additional evidence that the observed turning response is mediated by adenosine receptors. Moreover, the results suggest that A2A receptor activation is involved in adenosine-induced growth-cone turning of chicken sensory neurons. First investigations on the signal transduction chain of ADO-induced growth-cone turning It is well known that cAMP is an important downstream signal transducer of adenosine receptors [13]. Therefore, we tested the effect of the cAMP antagonist Rp-cAMPS [41] on adenosine-induced growth-cone turning. This membrane permeable antagonist was added in a concentration of 20 μM (data not shown) or 100 μM to the bath and pipette solution. Under these conditions the attractant response towards a pipette that contained 100 μM of ADO, in addition to the antagonist, was inhibited. Rp-cAMPS also blocked the attractant response towards a pipette filled with the specific A2A receptor agonist CGS 21680. In these experiments, the pipette contained 10 μM CGS 21680 and Rp-cAMPS was added to the bath and to the pipette solution in a concentration of 100 μM. Together these data suggest a contribution of cAMP to the signal transduction chain of adenosine-receptor-induced growth-cone turning (Fig. 4). Different downstream effectors of cAMP are known [42]. One important member of this group is PKA, which can be blocked by the specific inhibitor KT 5720 [43]. Here we tested the effect of KT 5720 on adenosine-receptor- mediated growth-cone turning. KT 5720 was added to bath and pipette solution in a concentration of 10 μM. Under these experimental conditions growth-cone turning towards a pipette that contained 100 μM of ADO was blocked significantly. However, under the same experimental con- ditions, KT 5720 failed to block the attractant response of a pipette filled with 10 μM of the A2A specific agonist CGS 21680 (Fig. 4). These seemingly inconsistent results reveal the need for a thorough investigation of the signal transduction chain. Discussion The present study demonstrates, for the first time, that extracellular ADO gradients can guide neuronal processes. Fig. 4 Effect of cAMP signalling on adenosine-receptor-mediated growth-cone turning. The cAMP antagonist Rp-cAMPS (100 μM) inhibits growth-cone turning induced by a 1-μm pipette filled with ADO (100 μM) or CGS 21680 (10 μM). Whereas the PKA inhibitor KT 5720 (10 μM) blocked the turning of growth cones in an ADO gradient, this was not the case when the A2A receptor selective agonist CGS 21680 was used as a chemoattractant. Displayed are the average turning angles, error bars indicate standard error of the mean (SEM), and n the sample size. Significant differences from the control group are marked (*P<0.05; **P<0.01; Mann-Whitney U test). Antagonists and inhibitors were added to the culture medium and pipette solution. Data of the control experiments and the ADO-induced turning have been taken from Fig. 3