Cgs 21680

The following drugs and reagents were purchased from Sigma Aldrich (St. Louis, MO, USA): (R)(−)-α-methylhistamine dihydrochloride, histamine dihydrochloride, adenosine deaminase (from bovine spleen), Percoll, quinpirole dihydrochloride. Dimaprit was from Axon MedChem (Reston, VA, USA). CGS-21680 was from Cayman Chemical (Ann Arbor, MI, USA). N-α-[methyl-³H]-histamine (78.3 Ci·mmol⁻¹) and CGS-21680-[carboxyethyl-³H (N)]-(35.2 Ci·mmol⁻¹) were from Perkin Elmer (Boston, MA, USA).

500 nM CGS 21680 (Cayman) or 100 μM adenosine (Sigma) were puff-applied onto the AIS or nodes of Ranvier. The drugs were added to aCSF with 100 μM Alexa 594 to monitor the spread of the drugs. 2-3 MΩ pipettes were filled with this solution and placed 10 μm away from the targeted axon. Positive pressure was applied either with a micro-injector (PMI-100, Dagan) for 20 ms at 10 psi or controlled manually with a syringe. In both cases, positive pressure was calibrated and live monitored to eject drug over a radius of 20 μm or less (from the tip of the pipette set at saturating intensity to the edge of detectable fluorescence). The flow of the perfusion was set to wash out the puffed drug in the direction away from the targeted neuron. The AIS was detected by adding 100 μM Alexa 594 into the intracellular solution and its diffusion into the axon was live monitored. The pipette puffing onto the AIS was placed near the distal AIS 25-30 μm away from the soma. The pipette puffing onto the nodes was placed near an axonal branch (detected from the Alexa 594 fill) or green GFP signal detected along the axon of a Thy1-Caspr-GFP mouse. Caspr was post hoc immunolabeled to confirm the presence of nodes at the puffing sites.