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The maturation of terminal arbors of retino-geniculate axons was studied in normal kittens from 1 to 8 postnatal weeks. Horseradish peroxidase injected into the optic tract rostral to the lateral geniculate nucleus gave a dense fill of cut axons and their terminals, resembling results obtained by the Golgi methods. At 1 and 2 weeks postnatal, the overall size and extent of axon arbors is not significantly different than in the adult. However, terminal branches of axon arbors at this age give rise to high variable endings. They terminate in finely divided sprays of finger-like extensions and filopodia bearing small spikes rather than the characteristic clusters and strands of crenulated terminals of adult axons. The bases of these sprays are broad and irregular in contour, with foliate growth-cone-like structures occurring at the ends of some branches. At 3 weeks postnatal, terminal swellings become thicker and more crenulated in contour. By 5 to 6 weeks, axon arbors have adult-like terminals with respect to their size, shape and arrangement, although entire branches may still be immature with irregular terminal swellings. Small slightly indented terminals are also seen for the first time. By 8 weeks, axon arbors are generally mature, but occasionally have a bizarre and immature arrangement of fine extensions or growth-cone-like tips. Although these observations do not establish whether reduction of branches or of terminals takes place during postnatal maturation, they demonstrate that kitten retino-geniculate axon terminal arbors are highly immature and undergo considerable changes during the period optimal for induction of sprouting by eye enucleation. The morphogenetic maturation of terminal branches that begins at 3 weeks also marks a decline in their sprouting capacity, even though remodeling of terminals is not complete until after 8 weeks of age.

Individual retino-geniculate axon arbors from kittens of 1 to 8 postnatal weeks were densely filled with horseradish peroxidase. The light-microscopical appearance of various types of immature growing tips was correlated with their synaptic relations as seen in the electron-microscope. During the first 2 postnatal weeks, the broad irregular bases of spray-like terminal extensions as well as foliate growth-cone-like terminals contain the round synaptic vesicles and pale mitochondria characteristic of adult retinal terminals. They form elementary glomerular arrangements in which they are the central profile synapsing with dendritic profiles. Many postsynaptic profiles contain large electron-lucent vesicles characteristic of growing neurites, whereas the immature retinal terminations do not. The more numerous finger-like growing tips also contain round synaptic vesicles and make short en passage contacts onto dendrites and outgrowths of perikarya of geniculate cells. The latter type of contact is not seen in the adult. After 3 weeks, retinal terminals are larger and make contacts in distinctly glomerular arrangements. Glial sheaths are evident for the first time. By 6-8 weeks, the terminals which appear crenulated in the light-microscope are always the central profile in a mature glomerulus. Smaller rounder terminals make simple axo-dendritic contacts as in the adult. The few immature terminal structures seen at 8 weeks also contain synaptic vesicles, but their contacts are not adult-like, nor are their synaptic arrangements entirely surrounded by glia. These findings, along with those of the previous paper, demonstrate that (a) many retino-geniculate terminals establish synaptic contacts long before attaining their adult form; (b) during the period optimal for induction of translaminar axon sprouting (1-2 weeks postnatal), only a few immature terminal forms participate in elementary glomeruli; (c) the decreasing malleability of these axons after 3 weeks is accompanied by an increase in crenulated terminals that enter glomeruli surrounded by glial sheaths. It is suggested that ensheathing of synaptic arrangements by astrocytic glia may be one factor which subsequently impedes axon sprouting in older animals.

Four large geniculate cells that were densely filled with horseradish peroxidase have been studied in detail, at first light microscopically, to define the shape and distribution of the dendritic arbors, and then electron microscopically, to define the pattern of synaptic contacts upon their surfaces. Three of the cells had the morphological characteristics of a class 1 cell, and one cell was intermediate between class 1 and 2. All of these cells had few of the characteristic grape-like appendages, thought to receive retinal input within synaptic glomeruli. Retinal afferents were mainly distributed around proximal juxtasomatic dendritic segments and dendritic branch points. Some retinal afferents made simple axodendritic contacts, while others formed a part of a synaptic glomerulus. None of the retinal afferents forming synapses near the perikaryon were involved in glomeruli. Within the glomeruli involving class 1 cells, retinal terminals can relate both to dendritic segments, particularly near branch points, as well as to dendritic appendages. The few singly placed grape-like appendages were always contacted by retinal afferents, but not necessarily in a glomerular arrangement. Terminals interpreted as cortico-geniculate were seen most commonly upon peripheral dendritic segments, while those containing pleomorphic vesicles (F) were distributed more or less evenly over all parts of the dendritic and perikaryal surface. The perikaryon itself was contacted by F-type terminals but was not contacted by retino-geniculate or cortico-geniculate terminals. A class of slender axonal terminal, containing round synaptic vesicles and few or no mitochondria, was found contacting two of the four perikarya, but the origin of these slender axons is unknown. It is concluded that the surfaces of geniculate class 1 cells can be separated into several functionally distinct zones on the basis of the synaptic contacts they receive.

A fundamental question in central nervous system development is the timing of synaptogenesis in relation to invasion of targets by afferent axons. A related question is how growth cones transform into synaptic terminals. These two aspects of axon maturation were examined in developing mouse cerebellum, by labeling single axons with horseradish peroxidase, to study their form and cytology, and by immunocytochemical staining of a synaptic vesicle antigen, synapsin I, a phosphoprotein found on synaptic vesicles in all mature CNS synapses. From embryonic day 16 to postnatal day 3, horseradish peroxidase-labeled afferent axons extend well into the cerebellum and have simple forms. At embryonic day 16, axon growing tips are synapsin I-negative. Synapsin I is first expressed at embryonic day 17, and by embryonic day 18, fibers are stained throughout the cerebellum. Synapsin I expression coincides with a general increase in synaptic specializations, although growing tips continue to have the cytology of growth cones. During the period that axons have primitive shapes, synapsin I is distributed throughout the terminal arbor, corresponding to the presence of small vesicles along neurite lengths, even at non-synaptic sites. After postnatal day 3, when synaptic terminals develop into stereotypic shapes and engage in characteristic synaptic relations, synapsin I is restricted to boutons. Thus, the synapse-specific protein synapsin I is expressed in fetal mouse brain, long before nerve endings have the structure and connections of adult brain. In cerebellar axons, the expression of this protein follows axon arrival, coincides with the appearance of elementary synapses, and accompanies the transformation of growing tips into stereotypic synaptic boutons. The time course of expression of synapsin I, a phosphoprotein that may be involved in synaptic efficacy, suggests that transmitter release may influence early axon-target cell interactions.