Ketjenblack

After 2P microscopy, we anesthetized the animal in 0.016% tricaine in Ringer solution modified for extracellular space preservation (63 mM NaCl, 63 mM cesium gluconate, 2.5 mM KCl, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 25 mM glucose, 2 mM CaCl₂ and 1 mM MgCl₂)^{17 (link)}, based on the principle of extracellular space preservation by cell-impermeable solutes^{54 (link)}. We removed the eyes with a piece of lasso-shaped tungsten wire and removed the skin covering the brain dorsally by first making a small incision caudal and dorsal of the brain using the electrochemically etched tip of a tungsten wire and then, through this incision, inserting the tungsten wire tip under the skin and carefully pulling upward and rostrally, pulling away the skin without touching the brain. We carefully removed any remaining skin flaps with forceps. Finally, we chemically fixed the larva with 2% glutaraldehyde and stained it with the reduced osmium/thiocarbohydrazide/osmium stain, aqueous uranyl acetate and lead aspartate^{8 (link)}.
To make the sample sufficiently conductive to allow imaging even the superficial, plastic-adjacent areas of the brain, we dispersed Carbon Black (2.5% w/v, Ketjenblack, AkzoNobel) in the epoxy^{55 (link)}.

First, 30% Pd supported on Vulcan-XC-72R (from Cabot, Boston, MA, USA), Ketjenblack (from AkzoNobel, Amsterdam, The Netherlands) and Ti suboxides (from Atraverda, Abertillery, Wales, UK) was prepared using the sulphite complex route [34 (link)]. An appropriate amount of support was ultrasonically dispersed in water for one hour and then mixed with Pd-sulphite acidic solution. Subsequently, H₂O₂ was added to decompose the sulphite complex with the formation of a colloidal suspension (PdO_x/support) after pH correction (5.5). Hence, the suspension was filtered, copiously washed with water and dried at 80 °C. The obtained powder was carbothermally reduced at 500 °C in an inert atmosphere (Ar) to form Pd/Vul and Pd/KB. Pd/Ti_nO_2n–1 was formed by the reduction of the dried powder, at room temperature (25 °C), in a 10% H₂–90% Ar atmosphere.