Identified neuromuscular junctions were followed in the sternomastoid muscle of living mice for several months by repeated staining with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4- Di-2-ASP; Magrassi et al., 1987). Overall terminal growth occurred at many endplates; however, the branching pattern of presynaptic arbors was largely unchanged, even after several months. The absence of significant remodeling over time was not a result of dye-staining, since sprouting was readily induced at residual motor endings by partial denervation. We conclude that--apart from overall growth--most neuromuscular junctions in the adult mouse are stable over intervals that represent a significant fraction of the animal's lifetime.

Different cerebellar phenotypes are generated according to a precise spatiotemporal schedule, in which projection neurons precede local interneurons. Glutamatergic neurons develop from the rhombic lip, whereas GABAergic neurons originate from the ventricular neuroepithelium. Progenitors in these germinal layers are committed toward specific phenotypes already at early ontogenetic stages. GABAergic interneurons are thought to derive from a subset of ventricular zone cells, which migrate in the white matter and proliferate up to postnatal life. During this period, different interneuron categories are produced according to an inside-out sequence, from the deep nuclei to the molecular layer (we show here that nuclear interneurons are also born during late embryonic and early postnatal days, after glutamatergic and GABAergic projection neurons). To ask whether distinct interneuron phenotypes share common precursors or derive from multiple fate-restricted progenitors, we examined the behavior of embryonic and postnatal rat cerebellar cells heterotopically/heterochronically transplanted to syngenic hosts. In all conditions, donor cells achieved a high degree of integration in the cerebellar cortex and deep nuclei and acquired GABAergic interneuron phenotypes appropriate for the host age and engraftment site. Therefore, contrary to other cerebellar types, which derive from dedicated precursors, GABAergic interneurons are produced by a common pool of progenitors, which maintain their full developmental potentialities up to late ontogenetic stages and adopt mature identities in response to local instructive cues. In this way, the numbers and types of inhibitory interneurons can be set by spatiotemporally patterned signals to match the functional requirements of developing cerebellar circuits.

We have evaluated the efficacy of 18 cationic mitochondrial dyes that, as a class, show some ability to stain living nerve terminals. Several of these agents provide excellent staining of neuromuscular junctions in a wide range of species. More detailed studies of the most effective of these dyes--4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di- 2-ASP)--indicate that it has no lasting effect on the structure or function of motor nerve terminals. As demonstrated in the accompanying paper (Lichtman et al., 1987; see also Lichtman et al., 1986), 4-Di-2- ASP can therefore be used to follow the configuration of identified motor terminals over arbitrarily long intervals.

All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to harbour an intracellular symbiont. The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships. To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M. darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches. Molecular phylogenetic analysis unambiguously placed all these bacteria among the flavobacteria-bacteroides, indicating that the endosymbiont of M. darwiniensis is the sister group to the cockroach endosymbionts examined. Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts's evolution. These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites. A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M. darwiniensis.

Animal cells are the sole habitat for a variety of bacteria. Molecular sequence data have been used to position a number of these intracellular microorganisms in the overall scheme of eubacterial evolution. Most of them have been classified as proteobacteria or chlamydiae. Here we present molecular evidence placing an intracellular symbiont among the flavobacteria-bacteroides. This microorganism inhabits specialized cells in the cockroach fat body and has been described as a mutualistic endosymbiont of uncertain phylogenetic position. The small subunit ribosomal DNA of these bacteria was analysed after polymerase chain reaction amplification to investigate their phylogeny. The endosymbionts of five species of cockroaches were found to make up a coherent group with no close relatives within the eubacterial phylum defined by the flavobacteria. In addition, the relationships among the endosymbionts, as revealed by DNA sequence data, appeared to be congruent with the host taxonomic relationships. Based on the host fossil record, a tentative calibration of the nucleotide substitution rate for the cockroach flavobacteria gave results congruent with those obtained for the aphid endosymbiotic proteobacteria.