Fig. 3. Sevoflurane does not affect the presynaptic secretory machinery. To test whether sevoflurane-induced suppression of synaptic transmission between visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1) was due to the perturbation of presynaptic release machinery, synapses were tested in either the absence or the presence of 3% sevoflurane (depresses the synapse almost completely) and the dye FM1-43. Neurons were paired overnight and allowed to develop synapses. Synapses developed at the contact site between the pairs, and VD4 processes were seen often unsheathing the LPeD1 somata ( arrows , A and D ). To demonstrate the pattern of dye localization in a control VD4, the cell was impaled intracellularly, and the dye was added to the preparation. VD4 was prevented from spiking by injecting hyperpolarizing current (0.2 nA) for 10 min, and images were acquired. No staining was discernable in VD4 ( B ). The presynaptic cell was then stimulated (100 action potentials) by current pulses, and the dye was replaced with normal, cold saline. Images were acquired again, which revealed punctate staining both at the contact site between the cells and also in VD4 processes encircling the LPeD1 somata ( arrows , C ). The above experiment was repeated in the presence of 3% sevoflurane ( D – F ). Sevoflurane at a concentration that blocks synaptic transmission almost completely also failed to prevent the uptake of the dye FM1-43, and punctate staining ( F ) similar to that seen under control ( C ) conditions was clearly discernable in the presence of this anesthetic.