The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

The physiological role of vesicular zinc at central glutamatergic synapses remains poorly understood. Here we show that mice lacking the synapse-specific vesicular zinc transporter ZnT3 (ZnT3KO mice) have reduced activation of the Erk1/2 MAPK in hippocampal mossy fiber terminals, disinhibition of zinc-sensitive MAPK tyrosine phosphatase activity, and impaired MAPK signaling during hippocampus-dependent learning. Activity-dependent exocytosis is required for the effect of zinc on presynaptic MAPK and phosphatase activity. ZnT3KO mice have complete deficits in contextual discrimination and spatial working memory. Local blockade of zinc or MAPK in the mossy fiber pathway of wild-type mice impairs contextual discrimination. We conclude that ZnT3 is important for zinc homeostasis modulating presynaptic MAPK signaling and is required for hippocampus-dependent memory.signal transduction | synaptic zinc | CA3 | cognition S ignals mediated by the Erk1/2 pathway of the MAPK family regulate a myriad of functions in the brain including development (1), memory stabilization (2), and drug addiction (3). Erk has been shown to regulate neuronal plasticity through direct actions on synaptic effectors (4, 5) or by altering gene expression (6, 7). Memory consolidation, reconsolidation, extinction, and persistence can all be impaired by Erk inhibition (8-12).The mossy fiber (MF) pathway of the hippocampus shows high levels of Erk activation under basal conditions (13). A feature of MF terminals is a high concentration of zinc in synaptic vesicles (14). Zinc is highly compartmentalized in neurons because of the coordinated actions of protein transporters and buffers (15). Zinc is coreleased with glutamate during MF exocytosis (16), and it can modulate ionotropic receptor currents (17-20) and synaptic plasticity in tissue slices (21-24). Exogenous application of zinc also can activate tyrosine kinase receptor B (TrkB) and Erk in neurons (21,25,26), but whether synaptic vesicle zinc modulates neuronal signal transduction is unclear. Little is known about the role of synaptic zinc in learning and memory. The zinc transporter ZnT3 is localized in clear synaptic vesicles of cortical glutamatergic terminals (27,28). Targeted deletion of ZnT3 prevents synaptic vesicle zinc uptake (29) and ablates the extracellular release of zinc by action potentials (16). Disruption of zinc homeostasis in ZnT3KO mice appears to be synapse specific, because zinc levels are reduced in fibers normally containing zinc but not in cell bodies or brain regions devoid of vesicular zinc (29,30). Further, seizureinduced increases in somatic (i.e., extrasynaptic) zinc are spared in ZnT3KO mice (31). Although initial attempts did not reveal cognitive deficits in ZnT3KO mice (32), recent evidence showed impaired fear memory (33) as well as accelerated aging-related decline of spatial memory (34) in ZnT3KO mice. Here we analyze the role of synaptic vesicle zinc for Erk-dependent signaling in the hippocampus and memory formation in young adult mice. ResultsZinc Is Requi...

Terrestrial vertebrates have evolved two anatomically and mechanistically distinct chemosensory structures: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Although it has been generally thought that pheromones are detected through the VNO, whereas other chemicals are sensed by the MOE, recent evidence suggests that some pheromones may be detected through the MOE. Odorant receptors in the MOE are coupled to the type 3 adenylyl cyclase (AC3), an enzyme not expressed in the VNO. Consequently, odorants and pheromones do not elicit electrophysiological responses in the MOE of AC3 Ϫ/Ϫ mice, although VNO function is intact. Here we report that AC3 Ϫ/Ϫ mice cannot detect mouse milk, urine, or mouse pheromones. Inter-male aggressiveness and male sexual behaviors are absent in AC3 Ϫ/Ϫ mice. Furthermore, adenylyl cyclase activity in membranes prepared from the MOE of wild-type mice, but not AC3 Ϫ/Ϫ mice, is stimulated by 2-heptanone, a mouse pheromone. We conclude that signaling through AC3 in the MOE is obligatory for male sexual behavior, male-male aggressiveness, and the detection of some pheromones.

Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN two days after training for contextual fear memory reduced contextual memory measured two weeks after training indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus.

Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.