Ramó n y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growthassociated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser 3 Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability. High-resolution imaging studies of altered nerve cell structure under the influence of synaptic input (1-3) provide a cellular basis for the view that learning involves structural modification of synapses (4, 5). One molecule that has been implicated in input-dependent alterations of synaptic morphology is the growth-associated GAP-43 protein (6), a protein kinase C (PKC) (7,8) substrate and an intrinsic determinant of structural change at the synapse. GAP-43, previously implicated in memory storage processes (9-16), binds to actin (17) and fodrin (18), and by such protein-protein interactions may affect morphological change.To determine whether the neuron-specific GAP-43 growth protein in fact regulates memory formation, we studied the effect on learning and synaptic potentiation of its overexpression in transgenic mice. The GAP-43-null mutation is lethal (19). Because evidence from this and other laboratories indicated that learning increases GAP-43 phosphorylation (9-16), one might expect that a transgenic mouse that overexpresses phosphorylatable GAP-43 would demonstrate enhanced learning. A critical corollary of this prediction is that such genetically enhanced learning would not occur if the PKC site of the overexpressed GAP-43 were mutated to prevent its phosphorylation. Materials and MethodsAnimals. Transgenic mice production has been described in detail elsewhere (20). Brief ly, to construct the expression cassette, an 8.2-kb EcoRI GAP-43 genomic fragment including the Thy-1.2 promoter was used. Germline-transmitting chimeras were obtained by standard injection into C57BL͞6 blastocysts, and the mutation was crossed into either C57BL͞6 or C2D2͞DBA genetic backgrounds. G-Phos is the S42wt line, G-NonP the S42A line, and G-Perm the S42D line. Nontransgenic, wild-type (WT) mice from the breeding program were used as controls. Transgenic animals were screened by slot blot hybridization.Slot Blot and in Situ Hybridization. Genomic DNA purified from mouse tail was used for slot blot hybridization by using a 32 P-labeled chick ...