DISCUSSIO N

The hypothesis that multiple diffusible growth-suppressant signals exist in the developing brain was tested by co-culturing explants from different brain regions in three-dimensional collagen gel matrices. The strategy was based on the fact that collagen gel matrices stabilize gradients of diffusible molecules (Lumsden and Davies 1983) and therefore make it possible to observe the effects of such factors released from one brain area on the neurite outgrowth of others. A variety of explants were co- cultured irrespective of their developmental relationships because the aim was to identify regions of the brain that secrete (and respond) to growth-suppressing activities. Four brain regions, cortex, superior colliculus, retina and septum were found to release activity which significantly reduced neurite outgrowth in other defined regions. The effects on neurite outgrowth from test explants were not due to a contact-dependent interaction since explants were positioned far enough apart to ensure that no contact between neurites from test explants and flanking explants occurred. Thus the neurite inhibitory effects can only be attributed to the secretion of diffusible factors from flanking explants. The inhibitory activities are tissue specific; cerebral cortical explants release an activity which, in culture, will inhibit septal and olfactory bulb neurites, but not those of the retina. Superior collicular explants release activity which also inhibits olfactory bulb neurites but has no effect on septal or retinal neurites. Indeed there is even a slight increase in the numbers of septal neurites. Septal explants also release activity which inhibits olfactory bulb axons (Pini 1993), but in contrast to superior collicular and cortical explants, it also inhibits retinal neurites. The fact that the inhibitory activities are tissue-specific not only argues the case for the existence of multiple diffusible inhibitory activities but also importantly eliminates the possibility that these inhibitory activities are simply non-specific toxic agents. If this were case, then neurite outgrowth from all test explants would be expected to be inhibited, this is not the case. In the isotypic control experiments described on p60, large flanking explants of cortex and septum had no effect on the outgrowth of the smaller centrally positioned explants. These observations add strongly to the case that the larger explants do not release non-specific toxic effects during culture. However, another possibility is that tissue-specific effects are due to a differential tolerance of neurites to toxic agents.

The inference here being that inhibition of neurite outgrowth will occur as consequence of a particular tissue having a low susceptibility to toxic agents. This possibility is unlikely since a single test explant which is inhibited by one particular tissue is not necessarily inhibited by all other neural regions that secrete growth-inhibitory activity. For example, retinal neurite outgrowth is inhibited by activity released by the septum but not by the cortex or superior collicular explants which both release inhibitory activity. This therefore rules out the possibility that a particular tissue, retina in this case, may possess a general innate low susceptibility to non-specific toxin.

The results indicate that there are multiple growth-suppressing signals in the embryonic brain but they do not directly indicate their numbers. However, it appears that there are at least three discernable activities. First, the cortex, septum (Pini 1993) and superior colliculus all inhibit the olfactory bulb and could therefore release identical activities. Secondly, only the septum inhibits the retina and so may release an activity distinct from those released by the cortex and superior colliculus. Thirdly, the cortex and superior colliculus could both release identical activities but since only the cortex inhibits the septum its activities may be distinct. On these data, the simplest though not necessarily complete scheme, would be that the cortex, septum and superior colliculus all release an identical activity which inhibits the olfactory bulb but in addition, the cortex and septum would each release separate inhibitory activities. In summary, while it is reasonable to suppose the existence of three distinct inhibitory activities, this may not be an upper limit. The septum secretes the most potent inhibitory activity which reduces retinal neurite outgrowth by 76% and cortical neurite outgrowth by 32%. This result would be consistent with the release by the septum of two separate activities with different potencies. Alternatively, such a difference could be indicating that the extent of inhibition simply reflects differences in the number or affinity of specific receptors to a single inhibitory factor secreted by the septum. Taken together with the results of Pini (1993) a simple flow diagram of the relevant tissue interactions is shown in figure 1.6.

FIGURE 1.6 - V C OLFACTORY BULB - V C • V C CORTEX SEPTUM SUPERIOR COLLICULUS -v e ■ V e RETINA SEPTUM

-ve denotes a significant in neurite growth inhibition, ± denotes no effect on neurite growth and +ve denotes a significant increase in neurite growth.

It is possible that the different inhibitory effects summarised in fig 1.6 could be due to different levels of secretion by the cortex, superior colliculus and septum of the same inhibitory activity. Since the septum but not the cortex or superior colhculus inhibits the retina, and all inhibit the olfactory bulb, it follows that the cortex and superior colliculus would have to secrete activity at low to subthreshold levels, while the septum would secrete at much higher levels. If so, it is unlikely that the cortex and superior colliculus would have potent effects on the olfactory bulb. However, this is not the case; the cortex releases activity which inhibits neurite outgrowth from olfactory bulb explants by 61% while the superior colliculus inhibits these neurites by 42%. In the experiments of Pini (1993) single septal explants reduced neurite outgrowth from olfactory bulb explants by 33.5%. If the potency of this effect were to double due to the positioning of a second septal flanking explant, then inhibition of the olfactory bulb outgrowth would be close to the level of inhibition described here for cortical explants (61%) and not far disimilar to that (42%) due to the superior colliculus. Moreover, the septum and retina exhibit mutual inhibition. Since single septal test explants are not inhibited by septal flanking explants, it follows that the mutual inhibition must be borne of distinct activities.

These results do not give any indication of the nature of the inhibitory activities or their mechanism of action. For instance it is not clear whether they inhibit neurite outgrowth by acting on growth cones directly, or haptotactically, that is, indirectly by binding to the collagen substratum. It is also unclear in these experiments whether in addition to their anti-trophic (growth inhibiting) effects these inhibitory activities have tropic (neurite-orientating) effects. It is likely that they do, in a similar fashion to the inhibitory activity secreted by the septum (Pini 1993) but determination of tropic effects has not been a primary objective of these experiments. In these co-culture experiments, test explants were almost completely surrounded by flanking explants, thus producing gradients with an entirely different shape that would exist with just one flanking explant.

Although the number of inhibitory activities is unknown, and further investigation is required to determine their molecular nature, it is clear from the results that there are multiple growth-suppressing signals present in the developing rat brain. These signals are secreted, diffusible and reduce neurite outgrowth from specific test explants in vitro.