The present study has shown that CSF from rat fetuses at different developmental ages (E17-E20) has a high protein concentration of around 3 mg/ml that declined to 2 mg/ml by E20. CSF when added to cultures of PC12 cells had different effects on survival, proliferation and neuronal differentiation depending on age. In contrast to the control groups, samples of CSF at all ages tested, gave a greater formazan absorbance reading indicating improved cell survival and/or proliferation. This was only significant with E18 CSF indicating a greater effect of CSF at this age. Interestingly we previously showed a greater proliferation of primary rat cortical cells in both E19 and E18 CSF over both controls and E17 or E20 CSF [
19]. The significant and most surprising result in the current study is the failure of PC12 cells to respond to E18 CSF with any measurable differentiation in either the MAP2 or beta III tubulin study, even though E18 CSF stimulated greater proliferation. These findings would fit with a three stage process, initial production of preplate neurons at E17, high rates of proliferation within the ventricular zone at E18, and migration of immature neurons and differentiation of migrated neurons within the cortical plate over E18-E20 [
45,
46]. The difference between the effects of PC12 cells and primary cortical cells indicate a possible difference between the pre-programmed development of the
in vivo neural stem/progenitor cells and the driving force of CSF composition in isolation.
In our experiments, we used cisternal CSF which is likely to contain both proliferation, differentiation and migration signals as it contains all the additions to CSF that are made as the fluid passes through the ventricles. It would of great value to test ventricular CSF and compare its effects since our previous arguments suggest that this may only contain proliferation and possibly differentiation signals but not migration signals [
6,
7,
23,
25]. Thus, one outcome of this study is that the parallel use of primary cortical cells and PC12 cells can help to elucidate the varying roles of developmental time-dependent programming of cells
versus the contributions made by the changes in CSF composition with age.
Almost 75% of CSF is secreted
in vivo by the CP located in the lateral ventricles with an additional 10% and 5% secreted by the CP in the third and fourth ventricles respectively [
47,
48]. Additional components are added to the ventricular CSF from the interstitial fluid of the brain parenchyma and from specific organs including the circumventricular organs. The most studied of these is the subcommissural organ which has been shown to be vital for particular physiological functions as well as keeping the CSF pathways open [
49‐
54]. Recent research demonstrates wa significant, if not major role for CSF in the survival, proliferation, migration and differentiation of neural stem/progenitor cells [
5,
11,
12,
19,
55]. Where problems exist in the CSF system, whether in flow or composition, this developmental program is adversely affected in ways that interfere in normal development [
4,
20,
34,
49,
56‐
61]. The most critical constituents of CSF are its protein components, the quality and quantity of which change during CNS development [
62‐
66]. About 20% of CSF proteins are derived from CNS, neurons, glial and leptomeningeal cells, whereas the remaining 80% originate from the blood or are synthesized by the CP [
30,
67]. The CP synthesizes and secretes many proteins, including various growth factors and neurotrophic factors into the CSF [
30,
67]. These proteins are carried by CSF bulk flow and provide the developing brain with trophic support for cell survival and neurogenesis [
68]. The utilization of neutralizing antibodies against growth factors in CSF showed that blocking of FGF2 in chick ECSF reduces cell proliferation, cell viability and neurogenesis in chick neuroepithelium [
69], while the thickness of the neuroepithelium and neuronal precursor proliferation decreased after anti-NGF antibody was injected into the fluid cavity of developing chick brain [
70,
71]. The current study provides a parallel cell line-based analysis system to that of primary brain cells to investigate the role of CSF. This approach is important as the interaction between primary cell age and CSF age has already been demonstrated [
19] while the isolated effect of CSF on a cell line might not expose all the interactions. The
in vitro survival, proliferation and neuronal differentiation of PC12 cells are dependent on certain growth factors which must also be present in the CSF to elicit the effects observed in this study. The evidence from previous studies indicates that understanding the detailed role of CSF in development, function and pathophysiology of the brain will be one key productive area to promote normal development and to develop strategies and treatments to prevent abnormal development and neuropathological conditions. Isolating CSF components responsible for these different effects can be achieved using the parallel approach we propose. In our recent work we have described a unique folate handling system serving the developing cerebral cortex that operates through the CSF [
26]. Obstruction to CSF flow or drainage results in a failure of cortical cells to release 10-formyl tetrahydrofolate, which we believe acts as a folate binding and transporter protein in CSF, and to an arrest in cell cycle and consequential deficient cortical development [
23,
24,
26]. Folate supply to the cerebral cortex can be affected independently of supply to the rest of the CNS and body and result in various cerebral folate deficiencies underlying a variety of neurological conditions that can be alleviated by specific folate supplements [
20,
22,
26,
57,
61,
72‐
81]. In addition to this folate supply to the developing cortex, there is a complex mix of growth factors and other important proteins in developmental CSF that are affected by CSF drainage and obstruction which remain to be tested for direct effects on the process of cortical development (unpublished data).