Spatial dynamics of the insulin receptor in living neurons.

ABSTRACT

Insulin signaling through the insulin receptor has long been studied in classic target organs, such as adipose tissue and skeletal muscle, where one of its effects is to increase glucose uptake. Insulin and insulin receptor are present in many areas of the brain, but the functions of brain insulin signaling outside feeding circuits are not well defined. It has been proposed that hippocampal insulin signaling is important for memory, that brain insulin signaling is deficient in Alzheimer's disease, and that intranasal insulin treatment improves cognition, but the mechanisms remain unclear and do not seem to involve increased glucose uptake by neurons. The molecular behavior of the insulin receptor itself is not well known in living neurons; therefore, we investigated the spatial dynamics of the insulin receptor on somatodendritic membranes of live rat hippocampal neurons in culture. Using single-molecule tracking of quantum dot-tagged insulin receptors and single-particle tracking photoactivation localization microscopy, we show that the insulin receptor is distributed over both dendritic shafts and spines. Using colocalization with synaptic markers, we also show that in contrast to the glutamate receptor subunit glutamate receptor subunit A1, the dynamics of the insulin receptor are not affected by association with excitatory synapses; however, the insulin receptor is immobilized by components of inhibitory synapses. The mobility of the insulin receptor is reduced both by low concentrations of the pro-inflammatory cytokine tumor necrosis factor α and by cholesterol depletion, suggesting an association with sphingolipid-rich membrane domains. On the other hand, the insulin receptor dynamics in hippocampal neurons are not affected by increased excitatory signaling. Finally, using real-time single-event quantification, we find evidence of strong insulin receptor exocytosis on dendritic shafts. Our results suggest an association of the neuronal insulin receptor with specific elements of the dendritic shaft, rather than excitatory synapses.