The development of new techniques and reagents has facilitated the detailed study and characterization of endocytic pathways. We used okadaic acid (OA), a naturally occurring toxin produced by marine dinoflagellates of the genus Prorocentrum, that is a potent and selective inhibitor of the serine/theorine protein phosphatase, phosphoprotein phosphatase 1, combined with a minimally invasive method to retrogradely label neurons and their processes in an adult spinal motor circuit. We demonstrate the prevalent expression of connexin 35 gap junction proteins and plaques at dendrodendritic excitatory mixed synapses between spinal motor neurons and interneurons. In addition, we discovered that OA appears to be entering spinal motor neurons through a clathrin‐independent internalization mechanism that we are currently investigating.Grant Funding Source: Supported by MH‐086994; NSF‐10 62963, and NSF‐0964114

The sense of position and movement of the paired and/or unpaired fins (“fin”, hereafter) is critical for executing rapid motor behavior in fishes. However, the location of the proprioceptive receptors involved in proprioception of fin movement is unknown; also unknown is whether or not afferent signals generated during fin movement are encoded to a “central body map” in the brain to reference the location of the fin in space. Here we provide results of work to describe and define the role of a mechanosensory receptor, the superficial neuromast, as a proprioceptive receptor in the male Western Mosquitofish, Gambusia affinis (“Mosquitofish”, hereafter). We sought to clarify whether or not superficial neuromasts associated with the male Mosquitofish sexually dimorphic fin serve as proprioceptive receptors for the proprioception of the fin to perform synchronous‐rapid‐rhythmic (SRR) movements essential for non‐cooperative coitus. In male Mosquitofish, we found that the number and distribution of the superficial neuromasts associated with the sexually dimorphic fin were significantly different (p = 2.6704 e‐05) from those found in female Mosquitofish. Removal of superficial neuromasts from the male resulted in significant impairment of SSR movements. These results suggest that the superficial neuromasts associated with the sexually dimorphic fin in the male Mosquitofish should be considered as a proprioceptive receptor.Grant Funding Source: Research supported by NSF‐1137725

Here we describe results of our work to develop a conductive resin and en bloc staining technique that together overcome the challenges of “charging” and “electron beam damage” posed by three‐dimensional (3D) electron‐based imaging methods such as serial‐section electron tomography. A conductive epoxy resin optimized for infiltration, immunocytochemistry, and uniform serial‐sectioning did not infiltrate evenly throughout the tissue and did not section well; however, it provided tissue section stability and eliminated tissue section charging, beam damage, and shrinkage. This led to our development of a new en bloc staining technique to use with the epoxy resin. Prior to embedding tissue in a non‐conductive resin, we made the tissue‐electron‐conductive and non‐charging. Together, the resin and the staining technique increase tissue contrast and electrical conductivity and overcome challenges posed by charging, radiation, and ion beam‐and electron‐induced surface contamination/artifacts. New tools such as the ones described here are needed to overcome the challenges of new technologies and take advantage of the increased resolution they provide. Supported by NS‐080687; GM‐108470; MH‐086994; NSF‐1062963, and NSF‐0964114.