Invitro application of gold nanoprobes in live neurons for phenotypical classification, connectivity assessment, and electrophysiological recording

Thermoregulatory neurons in the preoptic area of the anterior hypothalamus (POA) form synaptic networks, which affect responses that regulate body temperature. To characterize these pathways of activation, projections to effector control areas, like the dorsomedial hypothalamus (DMH), require labeling in live tissue slices. Traditional fluorescent dyes label axon terminals near an injection site, but unfortunately, also that of nearby fibers of passage. Here, we describe a novel methodology for retrograde labeling of neurons in vitro, which will allow for further electrophysiological recording. To determine if POA neurons project to the DMH, we have used nanometer-sized, gold nanoprobes, which provide for specific neuronal entry, via synapses in close proximity to the injection site. Upon neuronal entry, these nanoprobe complexes diffuse to the soma, where they are readily visualized and quantified. We found that conjugation of these gold nanoprobes with VGLUT-2 antibodies and polyethyleneimine (PEI) facilitates neuronal entry and a high level of labeling efficacy. This novel method, adapted from emerging cancer therapy technologies, is highly specific for determining axon terminal projections within particular neuronal populations, while maintaining neuronal viability for targeted live cell electrophysiological recording.