Correlation with basic differentiation processes of neurons

The development of the spinal cord involves the proliferation of
neurons, their migration to well-defined areas, fiber outgrowth and
synapse formation. The present study was designed to correlate the
spatiotemporal pattern of expression of synaptophysin, an integral
membrane protein of small synaptic vesicles, with these basic
processes occurring during the embryonic development of the rat
spinal cord. Thoracic segments of spinal cords from embryonic days
12, 14, 16, 18, 20 and of adult spinal cords were studied. S1
nuclease protection assays and immunoblots revealed minute amounts
of specific mRNA and synaptophysin at embryonic day 12. There was a
steep increase of mRNA between embryonic days 14 and 16, after
which levels reached a plateau. A rise in the amount of
synaptophysin in the spinal cord occurred between embryonic days 12
and 14, and the levels changed only slightly until the end of
embryonic development. Even higher levels of synaptophysin, found
in the adult spinal cord, may indicate that its biosynthesis
continued after birth. In situ hybridization histochemistry
revealed the localization of specific synaptophysin mRNA in the
neuroepithelium. However, immunocytochemistry failed to detect
synaptophysin in the neuroepithelial cells. Following migration of
the neuroblasts, synaptophysin was found in neurons concomitantly
with the onset of fiber outgrowth. Thus, already at embryonic day
12, outgrowing fibers of the dorsal root sensory neurons and of
motoneurons were synaptophysin positive. From embryonic day 14
throughout the prenatal period, strong synaptophysin
immunoreactivity was seen in the ventrolateral and dorsal parts of
the marginal layer. Most likely this staining pattern indicates
transient functional synaptic contacts because, in the adult spinal
cord, the corresponding region, the white matter, exhibited only
faint synaptophysin immunoreactivity. In the intermediate layer of
the embryonic spinal cord, which corresponds to the gray matter of
the adult spinal cord, synaptophysin-positive fibers were observed
prior to the formation of functional synapses. The latter are most
likely permanent, since synaptophysin in the adult spinal cord is
mainly confined to the gray matter. Our data (i) show transcription
and translation of synaptophysin within the neurons of the spinal
cord and correlate these processes with proliferation, migration,
fiber outgrowth and the formation of transient or permanent
synapses, and (ii) prove that synaptophysin is a marker for fiber
outgrowth in addition to synapse formation.