Correlated light and electron microscopic study of dopaminergic neurons and their synaptic junctions with DARPP-32-containing cells in three-dimensional reaggregate tissue culture

An antibody to tyrosine hydroxylase has been used in a correlated light and electron microscopic study to characterize dopaminergic neurons and synaptic junctions in three-dimensional reaggregate cell culture. Dissociated fetal mesencephalic cells containing dopamine neurons were coaggregated with dissociated fetal striatal cells in rotatory culture for 21 days. Sections of the coaggregates were stained by the peroxidase anti-peroxidase technique to reveal tyrosine hydroxylase-immunoreactive structures. Clusters of immunoreactive perikarya as well as dendrites and axons were observed. Immunolabeled perikarya were round or oval and approximately 20 microns in diameter. Boutons immunoreactive for tyrosine hydroxylase formed symmetric synapses, primarily with unlabeled dendritic shafts. Symmetric membrane specializations were also observed between tyrosine hydroxylase-positive boutons and unlabeled dendritic spines as well as with the perikaryon of an unlabeled medium-size neuron possessing a slightly indented nucleus. To characterize the neurochemical nature of the neurons postsynaptic to tyrosine hydroxylase-positive boutons in the reaggregates, an antibody against DARPP-32 (a dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein) and an antibody against tyrosine hydroxylase were employed to visualize striatal dopaminoceptive neurons and dopaminergic structures, respectively, in the same section. Examination of reaggregate sections at the light microscopic level demonstrated that DARPP-32-immunoreactive cells were distributed into discrete clusters that were associated with patches of tyrosine hydroxylase-positive axonal varicosities. Ultrastructural analysis of tyrosine hydroxylase-positive boutons in such clusters revealed that dopaminergic axons synaptically contacted DARPP-32-immunoreactive neurons as well as unlabeled neuronal structures.