Localization of the brain-specific high-affinity l-proline transporter in cultured hippocampal neurons: molecular heterogeneity of synaptic terminals.

The expression of a brain-specific, high-affinity Na+-(and Cl--)dependent l-proline transporter in subpopulations of putative glutamatergic pathways in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (Fremeau et al. , Neuron 8: 915-926, 1992). To assess further the cell-type and subcellular localization of PROT, we examined its distribution in low-density cultures of embryonic rat hippocampus. PROT immunoreactivity was detected beginning at 8 days in culture in a highly punctate pattern localizing to a subset of synaptic terminals. PROT was not detected at GABAergic terminals but was specifically localized to a subset of excitatory nerve terminals. PROT-labeled terminals showed partial apposition to AMPA-type and NMDA-type glutamate receptor clusters. Immunolabeling of isolated neurons grown in microisland cultures revealed that PROT was expressed by 60% of cultured hippocampal neurons. Individual microisland cultures were immunopositive for either PROT or glutamic acid decarboxylase, but never both. In the expressing pyramidal neurons, PROT was targeted to all presynaptic terminals. These findings indicate that PROT contributes to the molecular heterogeneity of glutamatergic terminals and suggest a novel presynaptic regulatory role for PROT in excitatory transmission at specific glutamatergic synapses.