Light and electron microscopic analysis of insulin binding sites on neurons in dissociated brain cell cultures

The distribution of insulin binding sites on primary cultured neurons and glia from the fetal rat was examined by the immunoperoxidase method using a specific insulin receptor antiserum. Light and electron microscopic analysis revealed a homogenous distribution of insulin binding sites on selective neuron-like cells of the dissociated cell culture system. To determine the influence of medium insulin on the distribution of insulin binding sites, dissociated cell cultures were maintained in the presence or absence of porcine insulin for varying time periods. We observed a significant increase in the number of insulin stained neuron-like cells maintained in insulin free defined medium compared to neuron-like cells maintained in insulin supplemented defined medium. Further, we examined the distribution of insulin binding sites after incubation with the antibody, which has agonistic properties in peripheral tissues, for varying time periods prior to fixation. Under these conditions, the light microscopic analysis revealed a heterogeneous (patchy) distribution of immunoreactive insulin binding sites, suggesting that the ligand receptor complex migrates. These results demonstrate the presence and distribution of insulin binding sites on neurons maintained in vitro, and provide morphological evidence to support a functional role for insulin in CNS tissues.