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 ...

The formation of CNS circuits is characterized by the coordinated development of neuronal structure and synaptic function. The activity-regulated candidate plasticity gene 15 (cpg15) encodes a glycosylphosphatidylinositol (GPI)-linked protein whose in vivo expression increases the dendritic arbor growth rate of Xenopus optic tectal cells. We now demonstrate that tectal cell expression of CPG15 significantly increases the elaboration of presynaptic retinal axons by decreasing rates of branch retractions. Whole-cell recordings from optic tectal neurons indicate that CPG15 expression promotes retinotectal synapse maturation by recruiting functional AMPA receptors to synapses. Expression of truncated CPG15, lacking its GPI anchor, does not promote axon arbor growth and blocks synaptic maturation. These results suggest that CPG15 coordinately increases the growth of pre- and postsynaptic structures and the number and strength of their synaptic contacts.

Hippocampal granule cells do not normally express the axonal growth- and plasticity-associated protein F1/GAP-43 in the adult rat. Using three different methods that lead to hypersynchronous activity in limbic circuits, expression of F1/GAP-43 mRNA can be induced in granule cells which is followed by sprouting in mossy fibers, the axons of granule cells. F1/GAP-43 mRNA expression in granule cells was induced in the temporal, but not septal, hippocampus beginning at 12 hours after kainic acid (KA) subcutaneous injection (10 mg/kg). Beginning 2 days after KA treatment, mossy fiber sprouts restricted to the temporal hippocampus were observed in the supragranular layer. In the same animal we also observed that levels of protein F1/GAP-43 immunoreactivity in this layer apparently increased at this same 2 day time point and same ventral hippocampal location. F1/GAP-43 protein levels and mossy fiber sprouting showed an increase up to 10 days after KA treatment. Sprouting was at a maximum at 40 days, the longest time point studied. These events parallel axonal regeneration with one critical difference: granule cell axons are not damaged by kainate. The rapid onset of axonal growth in the adult is striking and occurs earlier than reported previously (2 days vs. 12 days). Such growth closely associated with elevated levels of protein F1/GAP-43 may occur as a result of a) reactive synaptogenesis caused by the availability of post-synaptic surface on granule cell dendrites at the supragranular layer, b) Hebbian co-activation of the post-synaptic granule cells and their presynaptic afferents, and c) loss of target-derived inhibitory growth factor.

Mechanisms controlling dendritic arbor formation affect the establishment of neuronal circuits. Candidate plasticity gene 15 (CPG15) is a glycosylphosphatidyl inositol (GPI)-linked activity-induced protein that has been shown to function as an intercellular signaling molecule that can promote the morphological and physiological development of the Xenopus retinotectal system. A thorough understanding of CPG15 function requires knowledge of the spatiotemporal expression of the endogenous protein. We therefore cloned Xenopus cpg15 and used RNA in situ hybridization and immunohistochemistry to determine the pattern of CPG15 expression. cpg15 mRNA and CPG15 protein are first detectable in the developing spinal cord and become widespread as development proceeds. CPG15 is expressed in sensory regions of the brain, including the visual, auditory, and olfactory systems. Within the retina, CPG15 is only expressed in retinal ganglion cells. CPG15 protein is concentrated in axon tracts, including retinal axons. These data support a model in which CPG15 expressed in retinal ganglion cells is trafficked to retinal axons, where it modulates postsynaptic dendritic arbor elaboration, and synaptic maturation.

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The intricate circuitry of the nervous system has been shown to be refined by activity-dependent processes often involving the glutamate N-methyl-D-aspartate (NMDA) receptor. NMDA receptor activity has been directly associated with axonal growth during development and in adult models of synaptic plasticity. The axonal growth-associated protein GAP-43 has been involved in the same processes as the NMDA receptor, but a direct link between the two has never been demonstrated in vivo. It is attractive to think that the NMDA receptor may regulate axonal growth through GAP-43. We tested this idea in outgrowing axons of hippocampal granule cells, the mossy fibers. Granule cells normally only express GAP-43 in an organized outside-in manner during a restricted period in postnatal development paralleling the pattern of axonal extension. Here, we show that during postnatal development in a transgenic mouse bearing a GAP-43 promoter/lacZ reporter construct, granule cells also display an outside-in pattern of promoter activation as indexed by transgene expression (PATE). In fact, PATE precedes axonal outgrowth with temporospatial fidelity. Since PATE deactivates on growth termination, the promoter may function as a switch for an intrinsic program of regulated axonal growth. The NMDA receptor antagonist MK-801 administered within a restricted time frame (4-8 days) results in a decrease in the extent and intensity of mossy fiber staining. While levels of GAP-43 mRNA are significantly reduced in granule cells, GAP-43 PATE is not. The level of GAP-43 expression and axonal growth during development appears to be dually controlled by a transcriptional program that is activity-independent and by a posttranscriptional mechanism that is activity-dependent and NMDA mediated.

CPG15 (aka neuritin) is an activity-induced GPI-anchored axonal protein that promotes dendritic and axonal growth, and accelerates synaptic maturation in vivo. Here we show that CPG15 is distributed inside axons and on the axon surface. CPG15 is trafficked to and from the axonal surface by membrane depolarization. To assess CPG15 trafficking in vivo, we expressed an ecliptic pHluorin (EP)-CPG15 fusion protein in optic tectal explants and in retinal ganglion cells of intact Xenopus tadpoles. Depolarization by KCl increased EP-CPG15 fluorescence on axons. Intraocular kainic acid (KA) injection rapidly increased cell-surface EP-CPG15 in retinotectal axons, but coinjection of TTX and KA did not. Consistent with this, we find that intracellular CPG15 is localized to vesicles and endosomes in presynaptic terminals and colocalizes with synaptic vesicle proteins. The results indicate that the delivery of the neurotrophic protein CPG15 to the axon surface can be regulated on a rapid time scale by activity-dependent mechanisms in vivo.

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