Oxytocin is an essential hormone for mammalian labor and lactation. Here, we show a new function of oxytocin in causing plastic changes in hippocampal synapses during motherhood. In oxytocin-perfused hippocampal slices, one-train tetanus stimulation induced long-lasting, long-term potentiation (L-LTP) and phosphorylation of cyclic AMP-responsive element binding protein (CREB), and MAP kinase inhibitors blocked these inductions. An increase in CREB phosphorylation and L-LTP induced by one-train tetanus were observed in the multiparous mouse hippocampus without oxytocin application. Furthermore, intracerebroventricular injection of oxytocin in virgin mice improved long-term spatial learning in vivo, whereas an injection of oxytocin antagonist in multiparous mice significantly inhibited the improved spatial memory, L-LTP and CREB phosphorylation. These findings indicate that oxytocin is critically involved in improving hippocampus-dependent learning and memory during motherhood in mice.

Calcineurin and calpain, a Ca2؉ /calmodulin-dependent protein phosphatase and a Ca 2؉ -dependent cysteine protease, respectively, mediate neuronal cell death through independent cascades. Here, we report that during neuroexcitotoxicity, calcineurin A (CnA) is directly cleaved by calpain in vitro and in vivo, resulting in the enzyme being converted to an active form. Mass spectrometry identified three cleavage sites in CnA, two of which were constitutively active forms. Overexpression of the cleaved CnA induced caspase activity and neuronal cell death. Calpain inhibitors and membranepermeable calpastatin peptides not only blocked the cleavage of CnA, but also protected against excitotoxic neuronal cell death in vitro and in vivo. These results indicate that CnA is a crucial target for calpain, and the calpain-mediated activation of CnA triggers excitotoxic neurodegeneration. This study established a molecular link between calpain and calcineurin, thereby demonstrating a new mechanism for proteolytical regulation of calcineurin by calpain in response to certain pathological states.

Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase with close structural homology to the mitotic Cdks. The complex of Cdk5 and p35, the neuron-specific regulatory subunit of Cdk5, plays important roles in brain development, such as neuronal migration and neurite outgrowth. Moreover, Cdk5 is thought to be involved in the promotion of neurodegeneration in Alzheimer's disease. Cdk5 is abundant in mature neurons; however, its physiological functions in the adult brain are unknown. Here we show that Cdk5/p35 regulates neurotransmitter release in the presynaptic terminal. Both Cdk5 and p35 were abundant in the synaptosomes. Roscovitine, a specific inhibitor of Cdk5 in neurons, induced neurotransmitter release from the synaptosomes in response to membrane depolarization and enhanced the EPSP slopes in rat hippocampal slices. The electrophysiological study using each specific inhibitor of the voltage-dependent calcium channels (VDCCs) and calcium imaging revealed that roscovitine enhanced Ca2+ influx from the P/Q-type VDCC. Moreover, Cdk5/p25 phosphorylated the intracellular loop connecting domains II and III (L(II-III)) between amino acid residues 724 and 981 of isoforms cloned from rat brain of the alpha1A subunit of P/Q-type Ca2+ channels. The phosphorylation inhibited the interaction of L(II-III) with SNAP-25 and synaptotagmin I, which were plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins and were required for efficient neurotransmitter release. These results strongly suggest that Cdk5/p35 inhibits neurotransmitter release through the phosphorylation of P/Q-type VDCC and downregulation of the channel activity.

Proteins and peptides have been demonstrated to penetrate across the plasma membrane of eukaryotic cells by protein transduction domains. We show that protein transduction by 11 arginine (11R) is an efficient method of delivering proteins into the neurons of brain slices. Here, we demonstrate that PKA inhibitory peptide, fused with 11R and nuclear localization signal, delivers the peptide exclusively into the nuclear compartment of neurons in brain slices. This inhibitory peptide blocked both cAMP responsive element-binding protein phosphorylation and long-lasting long-term potentiation (LTP) induction, but not early LTP. These results highlight transduction of proteins and peptides into specific neuronal subcellular compartments in brain slices as a powerful tool for studying neuronal plasticity.