In the present study we have identified the extracellular matrix protein agrin as a major heparan sulfate proteoglycan (HSPG) in embryonic chick brain. Using monoclonal antibodies and a polyclonal antiserum to the core protein of a previously identified HSPG from embryonic chick brain, our expression screened a random-primed E9 chick brain cDNA library. Twelve cDNAs were isolated that were shown to be identical to the chick extracellular matrix protein agrin. Western blot analysis and immunocytochemistry confirmed that agrin is a HSPG that is identical with the HSPG from embryonic chick brain. A polyclonal antiserum to recombinant agrin protein recognized agrin as a diffuse band of over 400 kDa in extracts from brain and vitreous humor. The agrin immunoreactivity on the blot was shifted to a defined band of approximately 250 kDa after treatment of the samples with heparitinase or nitrous acid, and this banding pattern was indistinguishable from immunoreactivity obtained with antibodies to the brain HSPG. We also show that agrin binds tightly to anion exchange beads, indicating that the molecule is highly negatively charged, which is a hallmark of all proteoglycans. Furthermore, the agrin antiserum recognizes the affinity purified HSPG from chick brain and vitreous humor. Immunocytochemistry demonstrated that agrin is expressed in developing brain, and is especially abundant in developing axonal tracts, in a distribution identical to the staining of the brain HSPG with monoclonal antibodies. We also show that the anti-HSPG antibodies stain the synaptic site of the neuromuscular junction, in agreement with agrin expression. Thus, our studies demonstrate that chick agrin is a HSPG that is prominent in the embryonic chick brain. Since previous studies from our laboratories have shown that this proteoglycan interacts with neural cell adhesion molecule, our studies raise the interesting possibility that neural cell adhesion molecule and agrin are interactive partners that may regulate a variety of cell adhesion processes during neural development, including synaptogenesis.

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.

Fast excitatory synaptic transmission through vertebrate autonomic ganglia is mediated by postsynaptic nicotinic acetylcholine receptors (nAChRs). We demonstrate a unique postsynaptic receptor microheterogeneity on chick parasympathetic ciliary ganglion neurons-under one presynaptic terminal, nAChRs and glycine receptors formed separate but proximal clusters. Terminals were loaded with [3H]glycine via the glycine transporter-1 (GlyT-1), which localized to the cholinergic presynaptic terminal membrane; depolarization evoked [3H]glycine release that was calcium independent and blocked by the GlyT-1 inhibitor sarcosine. Ganglionic synaptic transmission mediated by nAChRs was attenuated by glycine. Coexistence of separate clusters of receptors with opposing functions under one terminal contradicts Dale's principle and provides a new mechanism for modulating synaptic activity in vivo.

A novel agrin isoform was identified based on the isolation of an agrin cDNA from E9 chick brain that lacked 21 base pairs (bp) in the NH2-terminal encoding region of the agrin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) of E9 chick brain mRNA confirmed the existence of this agrin isoform in brain, although the novel splice variant represents a minor fraction of agrin mRNA in brain. However, upon analysis of chick brain astrocyte mRNA, smooth muscle mRNA, and cardiac muscle mRNA by RT-PCR, we show that this novel agrin isoform is the predominant agrin isoform in these non-neuronal cell populations. We extended our analyses to examine the expression of this agrin mRNA isoform during chick development and show that the agrin mRNA lacking this 21-bp exon is up-regulated with brain development, consistent with the increase in glial number during brain development, while the agrin isoform that does not undergo splicing and thus contains the 21-bp exon is down-regulated in brain development. Because the 21-bp exon is inserted in the region of chick agrin which encodes the putative signal sequence of agrin, with the signal peptidase site immediately preceding the putative first amino acid of the mature protein being deleted as a result of splicing, these data raise the interesting possibility that the presence or absence of this alternatively spliced exon may differentially regulate processing of the agrin protein in neuronal and non-neuronal cells, respectively.