Correlative data are presented here on the developmental history, dynamics, histochemistry, and fine structure of intranuclear rodlets in chicken sympathetic neurons from in vivo material and long-term organized tissue cultures . The rodlets consist of bundles of -70 t 10 A proteinaceous filaments closely associated with -0 .4-0 .8 µ spheroidal, granulofibrillar (gf) bodies of a related nature . These bodies are already present in the developing embryo a week or more in advance of the rodlets . In early formative stages rodlets consist of small clusters of aligned filaments contiguous with the gf-bodies. As neuronal differentiation progresses these filaments increase in number and become organized into well-ordered polyhedral arrays . Time-lapse cinemicrography reveals transient changes in rodlet contour associated with intrinsic factors, changes in form and position of the nucleolus with respect to the rodlet, and activity of the gf-bodies . With the electron microscope filaments may be seen extending between the nucleolus, gf-bodies, and rodlets ; nucleoli display circumscribed regions with fine structural features and staining reactions reminiscent of those of gf-bodies . We suggest that the latter may be derivatives of the nucleolus and that the two may act together in the assemblage and functional dynamics of the rodlet . The egress of rodlet filaments into the cytoplasm raises the possibility that these might represent a source of the cell's filamentous constituents .

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Synapse formation and development were studied in organized cultures of mouse hypothalamic tissues from perinatal stages through a high degree of maturation during several months in vitro. Original explants from fetal and newborn animals were found to contain few definitive primordial synapses (ca. 4 per 10 grid-squares); newly-formed synaptic configurations began to appear with regularity in explanted tissues after the third day in vitro. These "immature" synapses contain small clusters of ,-300-600 agranular vesicles adjoining the presynaptic membrane, are usually devoid of mitochondria, and display diminutive junctional areas as yet little specialized. They are principally axodendritic, and may occur in the company of short, symmetrical, avesicular membrane-junctions which link neuritic extensions.By about one month in vitro a substantial increase in number of synapses (up to 25/grid square) as well as advances in degree of maturity are evident in the outgrown neuropil; axosomatic synapses are relatively less numerous, as in. vivo. Synaptic junction complexes henceforth appear well developed, and the presynaptic portions of boutons terminaux and boutons en passant now contain mitochondria and numerous agranular vesicles (sometimes packed in crystalloid arrays). Glomerulus-like profiles, synaptic spines displaying typical spine apparatuses, and multifarious synaptic complexes containing a variety of M 500-1,400 B granular vesicles are also in evidence. The impressive amount of synapse formation and degree of specialization observed here suggests the utility of such cultures as a model system for further hypothalamic investigations.Development of definitive synapses has been observed in long-term tissue cultures derived :from various regions of the avian and mammalian neuraxis (Bunge, Bunge and Peterson, '67). Bioelectric activity indicating the existence of functional synaptic connections also has been demonstrated in cultures from several of these areas (Crain, Peterson and Bornstein, '68); the onset and increasing complexity of the evoked response (Crain and Peterson, '67) has been correlated with specific morphological parameters of synaptic development as ascertained by electron microscopy (Bunge et al., '67), in cultures of rodent spinal cord.While earlier light microscopic studies (Borghese, '54; Hild, '54) have shown that portions of the hypothalamus could be cultivated in vitro for varying periods, and that terminal boutons resembling synaptic bulbs could be observed in hypothalamus cultures after silver-staining (Kim, personal communication), we know of no prior electron microscopic reports on the development of synapses in cultured hypothalamus as neuronal differentiation and maturation progresses in isolation from the host. This communication documents a systematic electron microscope study of the emergence, development, and morphology of synapses in mouse hypothalamic tissues from perinatal stages through a high degree 1This study w a s supported by NIH award K6-GM-15,372 and grants NINDB-0...

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