Agrin is a large extracellular matrix protein that directs the accumulation of acetylcholine receptors at the neuromuscular junction. Recent evidence suggests that agrin may be involved in organizing synapses in the visual system as well. Focussing on the pathway that controls accommodation and pupilloconstriction, this study examined the temporal pattern of agrin expression with reference to the organization of cholinergic synapses between embryonic chick Edinger-Westphal and ciliary ganglion neurons. In situ hybridization with an S35-labeled agrin cRNA probe was used to characterize agrin expression in the Edinger-Westphal nucleus during development. Agrin mRNA was detected in the Edinger-Westphal nucleus at all time points studied, from embryonic day 7 (E7, Hamburger and Hamilton stage 31) through newly hatched chicks. Throughout this period, agrin mRNA expression in Edinger-Westphal neurons was lower than in nearby oculomotor and trochlear neurons, suggesting that cells projecting to neuronal targets may require less agrin than those projecting to muscle. Agrin mRNA expression in the Edinger-Westphal nucleus at E7, E8, E9, and E10 was significantly higher than at E12. The early appearance of agrin mRNA coincides with the period during which acetylcholine receptors are being organized on ciliary ganglion neurons, consistent with the possibility that agrin contributes to neuron-neuron synapse formation in this pathway.

The free living nematode, C. elegans is understood at a level of detail equalled by few other organisms, and much of the cell biology and sequence information is proving of considerable utility in the study of parasitic nematodes. Already, C. elegans provides a convenient vehicle for investigating anthelmintic drug action and resistance mechanisms. Among the ionotropic receptors, with their important roles in the behaviour and development of the organism, are targets for anthelmintics. The subunits of nicotinic acetylcholine receptors of C. elegans form a large and diverse multigene family. Members of this family are among the 11 genes associated with resistance to the anthelmintic drug levamisole.

The mammalian neuromuscular junction has been extensively studied by different methods to understand better the biological aspects of its normal development, ageing and pathological conditions, such as disorders of neuromuscular transmission. In the present report, a new technique is described that combines confocal microscopy with the use of a vital nerve terminal dye (4-Di-2-ASP) and rhodamine-alpha-bungarotoxin to stain postsynaptic acetylcholine receptors in the same endplate. Nerve terminals in the sternomastoid muscles of living adult mice were stained with 4-Di-2-ASP, which labels intracellular compartments of the nerve terminal containing mitochondria. Slides of these muscles were viewed by confocal microscopy and images were stored on magnetic optical discs. This procedure was compatible with subsequent acetylcholine receptor staining with rhodamine-α-bungarotoxin and observation under the confocal microscope. Classical features of the adult neuromuscular junction were displayed, such as the branched-pattern distribution of the nerve terminals and receptors and their complete colocalisation. In addition, nerve fibres from intramuscular nerve branches with their neighbouring cells, nuclei and muscle fibre striations could also be visualised. We conclude that the present technique can complement existing methods of investigation of nerve terminal anatomy and pathology, particularly where preservation of 3-dimensional relationships is required and intracellular disturbances involving mitochondrial organisation, such as ageing or other degenerative disorders, may be present.