Recent evidence has demonstrated that cotransmission from mammalian neurons is not uniquely achieved by costorage and corelease of transmitters and cotransmitters from single varicosities, but also by the concurrent release of mediators segregated in separate synapses of individual neurons. An important question to be addressed is whether neurons show defined patterns of segregation or whether this is a plastic feature. We addressed this question by exploring the segregation pattern of the classical sympathetic transmitters norepinephrine (NE) and acetylcholine (ACh) and the cotransmitter neuropeptide Y (NPY) in sympathetic ganglionic neurons cocultured with cardiac myocytes. Using antibodies against NPY and the vesicular NE and ACh transporters VMAT2 and vesicular acetylcholine transporter (VAChT), we investigated the effect of ciliary neurotrophic factor (CNTF) or long (three weeks) culture periods on the segregation of VMAT2, VAChT, and NPY to separate varicosities. We found that although ganglionic neurons showed cell body coexpression of all the markers examined after three days, VMAT2 was segregated from VAChT in 43% of the VAChT-positive varicosities. In contrast, VMAT2 was only segregated from NPY in 16.3% of the NPY-positive varicosities. Cotransmitter segregation and VAChT expression was potentiated by both CNTF and longer times in culture. We also found two types of varicosities: one was smaller and located further from neuronal somata, and the other was larger, proximal to neuronal somata and had a higher level of segregation. These data demonstrate segregation of classical transmitters in sympathetic neurons and plasticity of neurotransmitter segregation. Finally, we discuss a possible functional correlate of segregation in sympathetic neurons.
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