360 Fig. 1a–c Relation between pipette geometry and gradient shape. a, b Scanning electron microscopic images of the two compared pipette types. Scale bar in a and b 1 μm. c Line plot of the concentration gradients generated by the respective pipette type (upper line 3 μm pipette, lower line 1 μm pipette). The plot was extracted from images of a TIRF measurement, as described in the “Materials and methods” section. Measuring parameters were as follows: pipette solution: 1 mM carboxyfluorescein; pressure: 35 mm water column; time: 5 min after inserting the pipette distance of 5 μm and 60 nM at a distance of 50 μm from the pipette tip for small pipettes filled with 100 μM ADO. For the large pipette, the values at the corresponding positions and pipette solution were 1.7 μM and 150 nM. Possible ADO content of the cell culture medium is not included in those values. Growth-cone turning in ADO gradients In standard experiments, the distance between growth cone and pipette tip was 50 μm at the start. Neurites from control cells elongated at a mean speed of 76 ± 5.2 μm/h (SEM). With respect to these constraints, we limited the measuring period to 15 min to guarantee a minimum distance between growth cone and pipette tip throughout the experiment. The turning behaviour of populations of growth cones in the presence of micro-gradients of ADO or vehicle controls is shown in Fig. 2 by superimposing traces of individual trajectories of neurite extension. A 1-μm pipette filled with 100 μM ADO consistently induced a chemotrophic turning response toward the pipette over the investigated time period of 15 min. Statistical analysis revealed that this ADO-induced turning response is significant compared to a vehicle control. Contrary to this finding, we did not observe a significant turning response using a 3-μm pipette filled with 100 μM ADO (Fig. 2). This result can be explained by the fact that growth-cone turning happens only if the gradient shape satisfies certain cellular conditions [36]. To examine whether growth-cone attraction by an ADO source is mediated by adenosine receptors, the unspecific adenosine receptor agonist NECA [11] was used. This agonist has a higher affinity to the different ADO receptors than ADO itself [37, 38]. Therefore we reduced the pipette concentration of NECA by a factor of ten relative to the ADO concentration. The rationale behind this approach is Purinergic Signalling (2008) 4:357–364 that growth cones can detect a concentration gradient only if the respective receptors are not saturated with the ligand [17]. This experiment resulted in a significant turning of growth cones towards the pipette filled with 10 μM NECA. Data are shown together with the results of other ADO receptor agonists and antagonists in Fig. 3. The turning response towards a pipette filled with 100 µM ADO could be blocked by using the unspecific ADO receptor antago- nist CGS 15943 [39]. This antagonist was added both to the bath solution and to the pipette solution in a concentration of 10 μM. The experiments with unspecific ADO receptor agonists and antagonists indicated that the ADO-induced growth- cone turning is transduced by ADO receptors. As the A2B and the A3 receptors have only a low affinity to ADO (KD 5,100 and 6,500 nM respectively [38]), a significant participation of these receptors in growth-cone-turning experiments seems implausible as the ADO concentration in this experiments was less than 100 nM. From this point of view the A1 and A2A receptors seem to be better candidates, as the binding constants of these receptors are 70 nM (A1) and 150 nM (A2A) [38]. Therefore, we tested the effect of R-(-)-PIA, an A1 receptor specific agonist [37]. Addition of 2 µM to the pipette solution was ineffective in inducing growth-cone Fig. 2a–e Adenosine induces positive turning of growth cones. Image of a growth cone from a chick sensory neuron a before and b 15 min after insertion of a 1-μm pipette filled with 100 μM ADO. c, d Trajectories of neurites grown for 15 min in a gradient. The pipette solution contained either c 100 μM ADO or d 10 μl/ml DMSO for the control. The trajectories were aligned in such a way that growth cones started from the origin and grew in direction of the ordinate. The ADO gradient is visualised schematically. e Cumulative distribution of turning angles of the growth cones exposed to gradients produced by a 1-μm pipette filled with either control solution (diamonds) or 100 μM ADO (squares) and a 3-μm pipette filled with 100 μM ADO (triangles). Values at the ordinate refer to the percentage of neurites with a turning angle inferior or equal to the angle given at the abscissa. Under experimental conditions which induce growth-cone attraction, the curve is shifted to the right