Direct cellular peptidomics of supraoptic magnocellular and hippocampal neurons in low-density cocultures

Genomic and proteomic studies of brain regions of specialized function provide evidence that communication among neurons is mediated by systems of diverse chemical messengers. These analyses are largely tissueor population-based, whereas the actual communication is from cell-to-cell. To understand the complement of intercellular signals produced by individual neurons, new methods are required. We have developed a novel neuron-to-neuron, serum-free coculture approach that was used to determine the higher-level cellular peptidome of individual primary mammalian neurons. We isolated magnocellular neurons from the supraoptic nucleus of early postnatal rat and maintained them in serum-free low-density cultures without glial support layers; under these conditions, they required low-density cocultured neurons. Coculturing magnocellular neurons with hippocampal neurons permitted local access to individual neurons within the culture for mass spectrometry. Using direct sampling, we obtained peptide profiles for spatially distinct, identifiable neurons within the coculture. We repeatedly detected 10 peaks that we assign to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enables the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides.