358 able by the corresponding receptors. This directional information can be coded by the shape of a concentration gradient [17]. It has been shown that concentration gradients of adenosine exist in the nervous system under in vivo conditions [18]. Due to the action of ectonucleoti- dases [19], each cell that releases ATP can be assumed to be the centre of an adenosine gradient. Moreover, cellular release of adenosine has also been shown [20, 21]. Here we use sensory neurons from chicken dorsal root ganglia (DRG), a system that has been used by others in growth-cone-turning assays [14, 22–25] to investigate the effect of adenosine on growth-cone turning. We show that micro-gradients of adenosine (ADO) generated by a micro- pipette technique are capable of inducing a positive growth- cone turning response. The present data demonstrate that the turning response is adenosine receptor mediated, as it emanates from experiments with the unspecific adenosine receptor agonist NECA and the unspecific adenosine receptor antagonist CGS 15943. Further studies with the A2A selective adenosine receptor agonist CGS 21680 confirm this finding and, apart from that, indicate that A2A receptor activation can induce a positive turning response. In contrast to this we found no effect on growth-cone turning when the A1 receptor agonist R-(-)-PIA was used. The precise nature of the adenosine receptor(s) involved in mediating the adenosine- induced turning response will require further study. The role of cAMP during adenosine-receptor-induced growth-cone turning seems to be elusive. While the cAMP antagonist Rp- cAMPS or KT 5720, which is a protein kinase A (PKA) inhibitor, block growth-cone turning in ADO gradients, the turning in CGS 21680 gradients is not affected by KT 5720, whereas Rp-cAMPS is effective and blocks CGS 21680- induced turning. Materials and methods Cell culture and experimental procedures Glass-bottom dishes were used for cell culture and turning experiments. To achieve optimum growth conditions, the bottom was first coated overnight with 0.1 mg/ml poly-D- lysine. Finally, the dishes were coated with 0.25 μg/ml laminin (Hoffmann-La Roche, Basel, Switzerland) for 1 h at 37°C. DRGs were dissected from 11-day-old chicken embryos and cultivated without dissociation overnight. The cell culture medium consisted of 90% DMEM F12 with HEPES (Invitrogen, Karlsruhe, Germany), 10% FCS (Invi- trogen, Karlsruhe, Germany), 100 μg/ml streptomycin, 100 units/ml penicillin and was supplemented with 25 ng/ml 7 S NGF (Becton Dickinson, Franklin Lakes, NJ, USA) [26, 27]. Mineral oil [mouse embryo tested, light oil (neat), Sigma, St. Louis, MO, USA] was used during the turning Purinergic Signalling (2008) 4:357–364 experiments to cover the cell culture medium in order to avoid outgassing and evaporation. The whole setup used to measure growth-cone turning was heated to 37°C. Special care was taken to avoid temperature differences in the cell culture dish during the measurement to circumvent distur- bance of the concentration gradients due to convection. Antagonists were added to the bath solution 30 min before the start of the experiment and were also present in the pipette solution. Generation of micro-gradients Micro-pipettes were made from borosilicate glass capillary tubes (outer diameter 1.6 mm; wall thickness 0.336 mm; Hilgendberg, Malsfeld, Germany) using an electrode puller (DMZ Universal Puller, Zeitz Instruments, Munich, Germany). The pulling programs were optimised to produce pipettes with either 80–100 or 40–60 MΩ resistance corresponding to an inner tip diameter of 1 or 3 μm, respectively. All pipette solutions were filtered through a 0.2 μm membrane filter to avoid clogging the pipette opening. The pipette solution was ejected by the constant hydrostatic pressure of a 35 mm water column. Micro-pipettes were inserted through the mineral oil layer into the cell culture dish with the help of a micro-manipulator. By rotating the cell culture dish, the angle between the pipette and longitudinal axis of the investigated neurite was adjusted to 90°. The pipette tip was positioned at a distance of 50 μm from the growth cone and at a 45° angle with respect to the direction of neurite extension. Characterisation of micro-gradients The shapes of the micro-gradients were characterised by using micro-pipettes that were filled with a 1 mM solution of 5-(and-6)-carboxyfluorescein (mixed isomers). Growth- cone turning is induced by concentration differences near the surface of the culture substrate where the growth cones adhere. Therefore gradients were characterised by total internal reflection fluorescence (TIRF) measurements that limit the investigated layer in the z-direction to the evanescent field depth [28]. This corresponds to the space where gradient detection by the growth cone takes place. Measurements were performed using an inverted epifluor- escence microscope (Axiovert 200, Zeiss, Jena, Germany) equipped with a 100× oil immersion objective (α Plan Fluar 100×/1.45 oil/∞/0.17, Zeiss, Jena, Germany), a CDD camera (IMAGO-SVGA), a TIRF condensor (TILL-TIRF module), and a 488 nm laser (Sapphire 488–20, Coherent, Santa Clara, CA, USA). The TIRF measurements were calibrated by normalising the background-corrected images of the gradients with an image, in which the chamber was filled with the undiluted pipette solution.