Goldfish brains were fixed Pexidartinib supplier and sections were incubated simultaneously with rabbit polyclonal anti-Cx34.7 IL antibody (see Table S1) and mouse monoclonal anti-Cx35 (Chemicon MAB3043) antibody, incubated with Alexa Fluor 488-conjugated goat anti-rabbit and/or Alexa Fluor 594-conjugated goat anti-mouse secondary antibodies, and coverslipped using n-propyl gallate-based mounting media. Sections were imaged using an Olympus BX61WI confocal microscope. Image analysis was performed using Image J (National Institutes of Health [NIH]) and MetaMorph software. Colocalization of Cx35 and Cx34.7 was measured as the percentage of the area labeled for Cx35 that was also labeled for Cx34.7 and the converse. The

antibodies used are listed in Table S1 along with their species of origin, designation, epitope recognition, source, and characteristics of detection of either or both Cx34.7 and Cx35. Specimens were processed by single-replica FRIL and one additional specimen by double-replica SDS-FRIL (sodium dodecyl sulfate-digested fracture replica labeling, which we designate as DR-FRIL). For single-replica samples, a gold “index” grid (aka “Finder” grid) was bonded to the coated surfaces using Lexan plastic (polycarbonate plastic) dissolved in dichloroethane; the samples were thawed and “grid-mapped” by confocal microscopy,

with which the location of the M-cell lateral dendrite was determined. A 150-μm-thick find more slice of goldfish hindbrain containing a Lucifer Yellow-injected M-cell was cryoprotected and fractured at −105°C in a prototype JFD-2 freeze-etch machine equipped with a turbopump but lacking a liquid-nitrogen-cooled shroud. Sodium butyrate The opened double-replica “sandwich” was coated with 3–5 nm of carbon, 1.5 nm of platinum, and ∼20 nm of carbon. Surgical and recording techniques were similar to those described previously (Smith and Pereda, 2003 and Curti and Pereda, 2004). Intracellular recordings were obtained in vivo from the lateral dendrite; both current clamp and single-electrode voltage clamp techniques were employed. Individual VIIIth nerve afferents were penetrated,

either at the posterior VIIIth root during simultaneous recordings with the M-cell’s lateral dendrite or less often, intracranially, close to the dendrite. Junctional resistance in each direction was estimated following Devor and Yarom (2002). These estimates of the junctional resistance assume a simple two neuron model with passive membrane properties coupled directly by a single junction. Experiments were performed on Rin cells expressing Cx35 or Cx34.7 tagged with eYFP or DsRed, respectively. To adjust the concentration of intracellular free Mg2+, we used pipette solutions containing different concentrations of MgCl2 and EDTA and the web-based Maxchelator software (http://www.stanford.edu/∼cpatton/webmaxcS.htm) to calculate free ionic concentrations. We thank Michael V.L. Bennett and Peter Sterling for their comments on the manuscript.