The proportion of synaptic contacts occurring on dendrites as well as on dendritic growth cones and filopodia was determined from electron micrographs of developing mouse (C57BL/6J) spinal cord . Comparable areas of the marginal zone adjacent to the lateral motor nucleus were sampled from specimens on the 13th-16th days of embryonic development (E13-E16) . At the beginning of this period, synapses upon growth cones and filopodia comprise about 80% of the observed synaptic junctions, but this proportion decreases with developmental time so that in E16 specimens growth cone synapses account for slightly less than 30% of the synaptic population. Conversely, at E13, synapses upon dendrites comprise less than 20% of the total number of synapses, but increase with developmental time so that they account for about 65 % of the synaptic population of E16 specimens .

The early development of the disynaptic cutaneous reflex pathway in the brachial spinal cord of rat fetuses was investigated both light and electron microscopically. The spinal cord areas which contain the neurons (association interneurons and lateral motor nucleus neurons) involved in this reflex pathway were identified in both neurofibrillar (silver-stained) and semithin plastic embedded specimens. Equivalent areas were identified in adjacent ultrathin preparations for electron microscopy and micrographs of these sample areas were taken of specimens at embryonic days 13.5-19.5 as well as for postnatal day 4 and adult specimens. The relative volume of association and dorsolateral motor neuropils occupied by synaptic boutons in these micrographs was determined using stereological methods. The mean number of synaptic junctions and boutons per 100 pz was also determined for the two neuropil areas at each developmental stage. These analyses show that synaptic junctions and boutons occur in the dorsolateral motor neuropil before they can be found in association neuropil. Synapses within motor neuropil are first seen at embryonic day 13.5. Synaptogenesis in the dorsolateral motor neuropil is temporally correlated with the presence of numerous fibers which, in silver-stained specimens, can be seen to arise from the association interneurons and to course into the dorsolateral aspects of the motor nucleus. Synaptic junctions and boutons are not observed in the association neuropil until embryonic day 14.5 and this correlates well with the time when collaterals of the primary afferent fibers can first be observed penetrating into the dorsal half of the intermediate zone from the anlage of the dorsal funiculus. The precocious development of synaptic junctions in dorsolateral motor neuropil in comparison to the association neuropil continues throughout the developmental period examined. These data support a retrograde pattern of synaptogenesis in the early development of the spinal cutaneous reflex pathway. Association interneurons make synaptic contacts with motor neurons before synapses are formed between primary sensory neurons and association cells. This sequence is the reverse of the normal flow of nerve impulses through spinal reflex pathways.One of the major goals of developmental neurobiologists is to understand how specific connections are formed between neurons during ontogeny (c.f. Sperry, '58; Jacobson, '70; Gaze, '70). The spinal cord is considered to be a useful model for such investigations in the central nervous systems of "higher" vertebrates because neurons become synaptically linked into specific somatotopic relationships with the motor and sensory peripheries at stages of development when the spinal cord is still "simple" enough to allow for comprehen- 177 JAMES E. VAUGHN AND JUDITH A. GRIESHABERmorphological development of segmental neural circuits as revealed by neurofibrillar stains and the development of early reflex movements of the forelimbs. The general sequence of development in the rat ...

The embryonic development of reflex forelimb movements produced by cutaneous stimulation of the forepaw was examined in five inbred strains of the house mouse, Mus musculus. A quantitative electron microscopic study of synapse formation between the neurons that comprise the spinal cutaneous reflex arc was also carried out on specimens from three of the strains subjected to reflex testing. This investigation provides evidence that there is significant genetically-associated variability in the developmental timing of synapse formation within this disynaptic pathway and in the reflex behavior which it mediates. Specifically, it was found that C57BL/6J embryos had greater numbers of synaptic junctions in the reflex pathway at embryonic days 14-16, and they also showed reflex movements earlier than LP/J embryos. C57BL/6J embryos also showed a more rapid increase in the number of boutons during this embryonic period. CBA/CaJ embryos displayed a temporal pattern of development that differed from both C57BL/6J and LP/J. At E15, CBA/CaJ embryos were more similar to LP/J with regard to both reflex activity and synapse number, but by E16, CBA/CaJ values for both of these measures were more similar to C57BL/6J. On the basis of the data detailed in the text, we suggest that the strains differ in the following manner: C57BL/6J embryos develop boutons rapidly but appear to be relatively inefficient in the actual formation of synaptic junctions; CBA/CaJ embryos develop boutons at a slower rate than C57BL/6J but form synaptic junctions more efficiently; LP/J embryos develop boutons slowly and are also relatively inefficient in forming synaptic junctions. The genetic implications of--and some developmental processes which might be responsible for--the observed strain differences in the timing of synaptic development are discussed in the text. There was no detectable genetic variability of the basic sequence in which the neurons of the cutaneous reflex arc develop their synaptic connections. For all three strains examined, the data indicated that synaptic closure occurred in a retrograde sequence with respect to the direction that neurotransmission normally flows between the neurons of this pathway. This finding agrees with results obtained by other investigators from a number of diverse vertebrate species, and such a widespread lack of variability implies that a retrograde sequencing of synapse formation is involved in the development of specific neuronal connectivities.

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