To explore the role of protein kinase A (PKA) in the late phase of long-term potentiation (L-LTP) and memory, we generated transgenic mice that express R(AB), an inhibitory form of the regulatory subunit of PKA, only in the hippocampus and other forebrain regions by using the promoter from the gene encoding Ca2+/ calmodulin protein kinase IIalpha. In these R(AB) transgenic mice, hippocampal PKA activity was reduced, and L-LTP was significantly decreased in area CA1, without affecting basal synaptic transmission or the early phase of LTP. Moreover, the L-LTP deficit was paralleled by behavioral deficits in spatial memory and in long-term but not short-term memory for contextual fear conditioning. These deficits in long-term memory were similar to those produced by protein synthesis inhibition. Thus, PKA plays a critical role in the consolidation of long-term memory.

CREB is critical for long-lasting synaptic and behavioral plasticity in invertebrates. Its role in the mammalian hippocampus is less clear. We have interfered with CREB family transcription factors in region CA1 of the dorsal hippocampus. This impairs learning in the Morris water maze, which specifically requires the dorsal hippocampus, but not context conditioning, which does not. The deficit is specific to long-term memory, as shown in an object recognition task. Several forms of late-phase LTP are normal, but forskolin-induced and dopamine-regulated potentiation are disrupted. These experiments represent the first targeting of the dorsal hippocampus in genetically modified mice and confirm a role for CREB in hippocampus-dependent learning. Nevertheless, they suggest that some experimental forms of plasticity bypass the requirement for CREB.

In an attempt to improve behavioral memory, we devised a strategy to amplify the signal-to-noise ratio of the cAMP pathway, which plays a central role in hippocampal synaptic plasticity and behavioral memory. Multiple highfrequency trains of electrical stimulation induce long-lasting long-term potentiation, a form of synaptic strengthening in hippocampus that is greater in both magnitude and persistence than the short-lasting long-term potentiation generated by a single tetanic train. Studies using pharmacological inhibitors and genetic manipulations have shown that this difference in response depends on the activity of cAMPdependent protein kinase A. Genetic studies have also indicated that protein kinase A and one of its target transcription factors, cAMP response element binding protein, are important in memory in vivo. These findings suggested that amplification of signals through the cAMP pathway might lower the threshold for generating long-lasting long-term potentiation and increase behavioral memory. We therefore examined the biochemical, physiological, and behavioral effects in mice of partial inhibition of a hippocampal cAMP phosphodiesterase. Concentrations of a type IV-specific phosphodiesterase inhibitor, rolipram, which had no significant effect on basal cAMP concentration, increased the cAMP response of hippocampal slices to stimulation with forskolin and induced persistent long-term potentiation in CA1 after a single tetanic train. In both young and aged mice, rolipram treatment before training increased long-but not short-term retention in freezing to context, a hippocampus-dependent memory task.The second messenger, cAMP, and cAMP-dependent protein kinase A (PKA) have been implicated in short-and longlasting synaptic plasticity in Aplysia and in short-and longlasting behavioral learning in Aplysia and Drosophila (1, 2). Recently, convergent pharmacological and genetic evidence has also implicated the cAMP system in short-lasting longterm potentiation (LTP) at the mossy fiber-CA3 synapse of rodent hippocampus (3-6), and, strikingly, in the stronger longer-lasting intermediate and late phases of long-lasting LTP (L-LTP) that follow three to four trains of tetanic stimulation in all three hippocampal pathways: the perforant, the mossy fiber, and the Schaeffer collateral (CA3-CA1) (3,(7)(8)(9)(10)(11)(12)(13)(14). LTP is a well studied example of synaptic plasticity in mammals, thought to be a candidate cellular mechanism for mediating some forms of explicit hippocampus-dependent memory (15, 16). L-LTP has been of particular interest in regard to this behavioral correlation, because it is much more persistent than the short-lasting long-term potentiation that follows a single tetanic train (7,8,9). L-LTP persists as long as it has been observed, up to 29 hr in vitro, and depends at later time points not only on PKA activity but also on transcription and translation (3,6,8,9), much like behavioral long-term memory.The dependence of L-LTP, in hippocampal slices and behavioral memory, on PKA a...

Mice carrying a truncated form of cAMP-responsive element binding protein (CREB)-binding protein (CBP) show several developmental abnormalities similar to patients with Rubinstein-Taybi syndrome (RTS). RTS patients suffer from mental retardation, whereas long-term memory formation is defective in mutant CBP mice. A critical role for cAMP signaling during CREB-dependent long-term memory formation appears to be evolutionarily conserved. From this observation, we reasoned that drugs that modulate CREB function by enhancing cAMP signaling might yield an effective treatment for the memory defect(s) of CBP ؉/؊ mice. To this end, we designed a cell-based drug screen and discovered inhibitors of phosphodiesterase 4 (PDE4) to be particularly effective enhancers of CREB function. We extend previous behavioral observations by showing that CBP ؉/؊ mutants have impaired long-term memory but normal learning and short-term memory in an object recognition task. We demonstrate that the prototypical PDE4 inhibitor, rolipram, and a novel one (HT0712) abolish the long-term memory defect of CBP ؉/؊ mice. Importantly, the genetic lesion in CBP acts specifically to shift the dose sensitivity for HT0712 to enhance memory formation, which conveys molecular specificity on the drug's mechanism of action. Our results suggest that PDE4 inhibitors may be used to treat the cognitive dysfunction of RTS patients.

Many studies have used "reverse" genetics to produce "knock-out" and transgenic mice to explore the roles of various molecules in long-term potentiation (LTP) and spatial memory. The existence of a variety of inbred strains of mice provides an additional way of exploring the genetic bases of learning and memory. We examined behavioral memory and LTP expression in area CA1 of hippocampal slices prepared from four different inbred strains of mice: C57BL/6J, CBA/J, DBA/2J, and 129/SvEms-+ Ter? /J. We found that LTP induced by four 100-Hz trains of stimulation was robust and long-lasting in C57BL/6J and DBA/2J mice but decayed in CBA/J and 129/SvEms-+ Ter? /J mice. LTP induced by one 100-Hz train was significantly smaller after 1 hr in the 129/SvEms-+ Ter? /J mice than in the other three strains. Theta-burst LTP was shorter lasting in CBA/J, DBA/2J, and 129/SvEms-+ Ter? /J mice than in C57BL/6J mice. We also observed specific memory deficits, among particular mouse strains, in spatial and nonspatial tests of hippocampus-dependent memory. CBA/J mice showed defective learning in the Morris water maze, and both DBA/2J and CBA/J strains displayed deficient long-term memory in contextual and cued fear conditioning tests. Our findings provide strong support for a genetic basis for some forms of synaptic plasticity that are linked to behavioral long-term memory and suggest that genetic background can influence the electrophysiological and behavioral phenotypes observed in genetically modified mice generated for elucidating the molecular bases of learning, memory, and LTP.

Today, the clinical notion of 'memory disorder' is largely synonymous with 'Alzheimer's disease.' Only 50% of all dementias are of the Alzheimer's type though, and dementias represent only the more severe of all learning/memory disorders that derive from heredity, disease, injury or age. Perhaps as many as 30 million Americans suffer some type of clinically recognized memory disorder. To date, therapeutic drugs of only one class have been approved for the treatment of Alzheimer's disease. Fortunately, basic research during the past 25 years has begun to define a 'chemistry of brain plasticity,' which is suggesting new gene targets for the discovery of memory enhancers.

Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.

We have used a combined genetic and pharmacological approach to define the time course of the requirement for protein kinase A (PKA) and protein synthesis in long-term memory for contextual fear conditioning in mice. The time course of amnesia in transgenic mice that express R(AB) and have genetically reduced PKA activity in the hippocampus parallels that observed both in mice treated with inhibitors of PKA and mice treated with inhibitors of protein synthesis. This PKA- and protein synthesis-dependent memory develops between 1 hr and 3 hr after training. By injecting the protein synthesis inhibitor anisomycin or the PKA inhibitor Rp-cAMPs at various times after training, we find that depending on the nature of training, contextual memory has either one or two brief consolidation periods requiring synthesis of new proteins, and each of these also requires PKA. Weak training shows two time periods of sensitivity to inhibitors of protein synthesis and PKA, whereas stronger training exhibits only one. These studies underscore the parallel dependence of long-term contextual memory on protein synthesis and PKA and suggest that different training protocols may recruit a common signaling pathway in distinct ways.