Retrograde and anterograde tracing combined with transmitter identification and electron microscopy

Fiber tracts in the brain are formed by neurochemically heterogeneous neuron populations. To distinguish between the different neurons that contribute to a fiber tract it is necessary to combine anterograde and retrograde tracing techniques with immunocytochemistry. In this article, we describe two techniques which allow for the neurochemical identification of retrogradely labeled neurons and anterogradely labeled axons on the ultrastructural level. The identification of the neurotransmitter identity of retrogradely labeled neurons is achieved by combining retrograde Fluoro-Gold tracing with preembedding immunocytochemistry, while the neurotransmitter identity of anterogradely labeled axons can be revealed by combining anterograde Phaseolus vulgaris-leucoagglutinin (PHAL) tracing and postembedding immunostaining.