Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system

Synaptophysin is a protein of synaptic vesicles and may be demonstrated in tissue sections of human brain and spinal cord by immunocytochemistry using a monoclonal antibody. Synaptophysin immunoreactivity was studied in paraffin-embedded sections of the central nervous system (CNS) in 14 normal human fetuses and neonates ranging in age from 8 to 41 weeks gestation, and in three brains with heterotopic neurons or malformations. A progressive expression of synaptophysin is seen in axonal terminals within grey matter in various parts of the CNS, beginning in the ventral horns of the spinal cord and brainstem tegmentum at 12–14 weeks. In the cerebellum, the molecular layer shows a band of reactivity from 18 weeks; by term two parallel bands of synaptophysin are seen in the molecular layer and reactivity also is demonstrated in the Purkinje and internal granular layers. In the cerebral neocortex, the molecular zone has weak synaptophysin reactivity as early as 10 weeks, though reactivity is not detected in the deep layers of the cortical plate until 19 weeks and in layers 2–4 until 25 weeks gestation. Synaptophysin reactivity is strong at the surface of neurons but not detected in their somatic cytoplasm; coarsely beaded reactivity within the neuropil probably corresponds to synaptic vesicles in terminal axons. Similar granular synaptophysin reactivity is seen around heterotopic neurons in the subcortical white matter, in dysgenesis of the cerebellar cortex and in the residual anencephalic forebrain. Thermal intensification by heating the incubating solution in a microwave oven often enhances immunoreactivity because of more complete antigen retrieval and is recommended for tissue stored in formalin or in paraffin for long periods. Synaptophysin provides a useful tissue marker of synaptogenesis during normal development and in cerebral dysgeneses, and may provide useful correlations with functional imaging of the brain in living patients. Used in conjunction with other neuronal markers, the expression of synaptophysin in terminal axons of distant neurons, in temporal relation to the maturation of the neurons they innervate, may provide clues to the pathogenesis of epilepsy in early infancy.