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We describe a new mutation, flathead (fh), that arose spontaneously in an inbred colony of Wistar rats. The mutation is autosomal recessive, and the behavioral phenotype of fh/fh rats includes spontaneous seizures, tremor, impaired coordination, and premature death. A striking feature of the fh mutation is a dramatic reduction in brain size (40% of normal at birth). In contrast, no abnormalities are evident in the peripheral nervous system or in other tissues outside of the CNS. Although bromodeoxyuridine incorporation assays indicate that the rate of cell proliferation in the fh/fh cortex is similar to that of unaffected animals, in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling assays reveal a dramatic increase in apoptotic cell death beginning after embryonic day 16 (E16). At E18 there is a 20-fold increase in cell death in the ventricular zone of fh/fh neocortex, and at postnatal day 1 (P1), the number of apoptotic cells is still two times that of normal. However, by P8 the extent of cell death in fh/fh is comparable to that of unaffected littermates, indicating that the reduction in brain growth is caused by abnormally high apoptosis during a discrete developmental period. Late-developing structures such as the cerebellum, neocortex, hippocampus, and retina are most severely affected by the fh mutation. Within these structures, later-generated neuronal populations are selectively depleted. Together, these results suggest that the flathead gene is essential for a developmental event required for the generation and maturation of late-born cell populations in the brain.

Cultured cerebellar granule neurons die by apoptosis when switched from a medium containing an elevated level of potassium (K+) to one with lower K+ (5 mM). Death resulting from the lowering of K+ can be prevented by insulin-like growth factor (IGF-1). To understand how IGF-1 inhibits apoptosis and maintains neuronal survival, we examined the role of phosphoinositide 3-kinase (PI 3-kinase). Activation of PI 3-kinase has been shown previously to be required for NGF-mediated survival in the PC12 pheochromocytoma cell line. We find that in primary neurons, IGF-1 treatment leads to a robust activation of PI 3-kinase, as judged by lipid kinase assays and Western blot analysis. Activation of PI 3-kinase is likely to occur via tyrosine phosphorylation of the insulin receptor substrate protein. Treatment with two chemically distinct inhibitors of PI 3-kinase, wortmannin and LY294002, reduces PI 3-kinase activation by IGF-1 and inhibits its survival-promoting activity, suggesting that PI 3-kinase is necessary for IGF-1-mediated survival. Death resulting from PI 3-kinase blockade is accompanied by DNA fragmentation, a hallmark of apoptosis. Furthermore, neurons subjected to PI 3-kinase blockade can be rescued by transcriptional and translation inhibitors, suggesting that IGF-1-mediated activation of PI 3-kinase leads to a suppression of "killer gene" expression. In sharp contrast to IGF-1, elevated K+ does not activate PI 3-kinase and can maintain neuronal survival in the presence of PI 3-kinase inhibitors. Therefore, survival of granule neurons can be maintained by PI 3-kinase dependent (IGF-1-activated) and independent (elevated K+-activated) pathways.