We have developed a 3-D culture technique for analysing the function of novel genes (Blackshaw et al. 1998), isolated from a cDNA library enriched in upregulated sequences from regenerating Retzius neurons in Hirudo medicinalis (Korneev et al. 1998). We have used this culture system to assay the effects of antisense oligodeoxyribonucleotides to protein 4.1, found in this library, upon microglial cell migration and axonal outgrowth. Protein 4.1 is a structural protein, also implicated in neurotransmitter receptor targeting and RNA maturation.

Antisense 15-mer oligonucleotides against the 5Ì end of the leech 4.1 cDNA, together with sense and nonsense oligos, were added to ganglion cultures, at three concentrations (10 nM, 100 nM and 1 µM). After 10 days, both the total neurite outgrowth and the number of microglia migrating from each ganglion were measured, on an Ibas 400 image analysis system. Only neurites visible in the gel matrix under phase-contrast optics were measured. Data were analysed using the Mann-Whitney U test. At 10 nM, no significant effects on microglial number were seen, whilst at the two higher concentrations all oligos produced a significant reduction in number. 1 µM oligos all produced a similar, significant reduction in neurite length, but at 10 nM only the antisense and nonsense oligos produced significant reductions (to 75.1 and 78.5 % of controls, respectively).

To investigate any possible toxicity of the leech oligos, we used the PC12 rat phaeochromocytoma cell line, which exhibits neuron-like properties after differentiation. PC12 cells were differentiated with nerve growth factor and then cultured in the presence of the leech oligos for a further 10 days, when cell numbers were counted. At 1 µM, the antisense and nonsense oligos produced a significant reduction in cell survival (to 46.2 and 52.1 % of controls) whilst at 10 nM, no reduction in cell number was seen.

In conclusion, we have seen that antisense 4.1 oligos have an effect upon neurite extension and microglial migration. At 1 µM this effect is uniform, which may be due to their toxicity at this level. At 10 nM, only the antisense and nonsense oligos produced a significant reduction in neurite growth and cell migration. 10 nM may represent the optimal concentration for this technique. It is also possible that the nonsense oligo is interacting with another gene. As there little leech genome data available, we cannot currently exclude this possibility.This work is supported by an MRC Studentship to J.V. and by a HFSP grant to S.E.B.

Blackshaw, S.E., Arkison, S., Davies, J.A. & Holmes, D. (1998). J. Physiol. 509.P, 191P.

Korneev, S., Fedorov, A., Collins, R., Blackshaw, S.E. & Davies, J.A. (1998). Inv. Neurosci. 3, 185-192.