Axonal targeting of agrin in cultured rat dorsal horn neurons

Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was present in the somata and in the axons but not in dendrites within the sensitivity of the detection. This intra-axonal localisation of agrin could only be seen after permeabilization. Furthermore, agrin immunoreactive axons were found adjacent to gephyrin, the postsynaptic glycine receptor-associated protein. Altogether, our results suggest that, as established at the neuromuscular junction, agrin may be involved in pre- to postsynaptic interactions in the central nervous system.