Transient appearance of Ca2+ -permeable AMPA receptors is crucial for the production of repetitive LTP-induced synaptic enhancement (RISE) in cultured hippocampal slices.

We have previously shown that repetitive induction of long-term potentiation (LTP) by glutamate (100 µM, 3 min, three times at 24-hr intervals) provoked long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (repetitive LTP-induced synaptic enhancement). Here, we examined the role of Ca2+ -permeable (CP) AMPA receptors (AMPARs) in the establishment of RISE. We first found a component sensitive to the Joro-spider toxin (JSTX), a blocker of CP-AMPARs, in a field EPSP recorded from CA3-CA1 synapses at 2-3 days after stimulation, but this component was not found for 9-10 days. We also observed that rectification of AMPAR-mediated current appeared only 2-3 days after stimulation, using a whole-cell patch clamp recording from CA1 pyramidal neurons. These findings indicate that CP-AMPAR is transiently expressed in the developing phase of RISE. The blockade of CP-AMPARs by JSTX for 24 hr at this developing phase inhibited RISE establishment, accompanied by the loss of small synapses at the ultrastructural level. These results suggest that transiently induced CP-AMPARs play a critical role in synaptogenesis in the developing phase of long-lasting hippocampal synaptic plasticity, RISE.

Analysis of gene expression changes associated with long-lasting synaptic enhancement in hippocampal slice cultures after repetitive exposures to glutamate.

We have previously shown that repetitive exposures to glutamate (100 muM, 3 min, three times at 24-hr intervals) induced a long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (for repetitive LTP-induced synaptic enhancement). To investigate the molecular mechanisms underlying RISE, we first analyzed the time course of gene expression changes between 4 hr and 12 days after repetitive stimulation using an original oligonucleotide microarray: "synaptoarray." The results demonstrated that changes in the expression of synapse-related genes were induced in two time phases, an early phase of 24-96 hr and a late phase of 6-12 days after the third stimulation. Comprehensive screening at 48 hr after the third stimulation using commercially available high-density microarrays provided candidate genes responsible for RISE. From real-time PCR analysis of these and related genes, two categories of genes were identified, 1) genes previously reported to be induced by physiological as well as epileptic activity (bdnf, grm5, rgs2, syt4, ania4/carp/dclk) and 2) genes involved in cofilin-based regulation of actin filament dynamics (ywhaz, ssh1l, pak4, limk1, cfl). In the first category, synaptotagmin 4 showed a third stimulation-specific up-regulation also at the protein level. Five genes in the second category were coordinately up-regulated by the second stimulation, resulting in a decrease in cofilin phosphorylation and an enhancement of actin filament dynamics. In contrast, after the third stimulation, they were differentially regulated to increase cofilin phosphorylation and enhance actin polymerization, which may be a key step leading to the establishment of RISE.

Repetitive induction of late-phase LTP produces long-lasting synaptic enhancement accompanied by synaptogenesis in cultured hippocampal slices.

Long-term plasticity of synaptic transmission is assumed to underlie the formation of long-term memory. Although the cellular mechanisms underlying short-term plasticity have been analyzed in detail, the mechanisms underlying the transformation from short-term to long-term plasticity remain largely unrevealed. We propose the novel long-lasting phenomenon as a model system for the analysis of long-term plasticity. We previously reported that the repetitive activation of cAMP-dependent protein kinase (PKA) by forskolin application led to an enhancement in synaptic strength coupled with synaptogenesis that lasted more than 3 weeks in cultured rat hippocampal slices. To elucidate whether this long-lasting synaptic enhancement depended on the induction of long-term potentiation (LTP) or on the pharmacological effect of forskolin, we applied glutamate (Glu) and correlated its dose with the production of the long-lasting synaptic enhancement. When the dose of Glu was low (10, 30 muM), only transient excitation or early-phase LTP (E-LTP) was induced by a single application and no long-lasting synaptic enhancement was produced by three applications. When the dose was raised to 100 or 300 muM, late-phase LTP (L-LTP) was induced by a single application and long-lasting synaptic enhancement was produced by three applications. The Glu-produced enhancement was accompanied by an increase in the frequency (but not the amplitude) of miniature EPSC and the number of synaptic structures. The enhancement depended on the interval of repetition and protein synthesis immediately after the Glu applications. These results indicate that the repetitive induction of L-LTP, but not E-LTP or transient excitation, triggers cellular processes leading to the long-lasting synaptic enhancement and the formation of new synapses.

Ultrastructural features of hippocampal CA1 synapses with respect to synaptic enhancement following repeated PKA activation.

We reported previously that repeated activations, but not a single activation, of cyclic AMP-dependent protein kinase (PKA), led to a slowly developing (requiring approximately 1 week to develop) long-lasting (lasting > or = 3 weeks) enhancement of synaptic transmission efficiency in the organotypic slice culture of the rat hippocampus. It was accompanied by an increase in the number of synapses identified immunohistochemically. To answer the question of whether the "perforated synapse", which is known to occur transiently after the induction of long-term potentiation (LTP) in combination with the enlargement of postsynaptic density (PSD), is involved also in this slow/persistent synaptic enhancement, we examined the ultrastructural changes after the repeated activations of PKA. The answer was partially yes (occurrence of perforated synapses was increased) but partially no (the increase in the number of perforated synapses was not transient but persistent; mean apparent size of PSD did not increase). These results suggest that the mechanism of the slow/persistent synaptogenesis shares limited features with the mechanism of the quick/transient morphogenesis after LTP.

Long-lasting synapse formation in cultured rat hippocampal neurons after repeated PKA activation.

Recently, we reported that the repeated activation of cyclic-AMP-dependent protein kinase (PKA) in the rat hippocampus under tissue culture conditions induced the enhancement of excitatory postsynaptic potential (EPSP), which lasted more than 2 weeks and was accompanied by the formation of morphologically identifiable synapses. Here we examined whether an equivalent synapse formation is induced in dissociated cell cultures of rat hippocampal neurons. Brief (15-min) application of Sp-cAMPS (a membrane-permeable analog of cyclic AMP) induced an increase in the number of synaptic sites (identified by the apposition of immunocytochemically labeled pre- and postsynaptic structures). There were two types of increase: a short-lasting one that lasted less than 24 h after a single application of Sp-cAMPS, and a long-lasting one that lasted more than 2 weeks after repeated applications. The long-lasting increase in synaptic sites was dependent on the time and interval of application and was suppressed by Rp-cAMPS (a PKA inhibitor). The synapses were judged to be active based on the endocytosis of FM1-43, a fluorescent dye. Electron microscopy confirmed the increase in the number of synaptic ultrastructures. The present results show that the synaptogenesis induced by repeated PKA activation is reproducible in a neuronal network that is reconstituted under dissociated cell culture conditions. This experimental system, together with the synaptogenesis in the slice culture system described previously, serves as a good in vitro model for the analysis of the process of conversion from short-lasting plasticity (lasting for hours) into a long-lasting one (lasting for days-weeks).

Non-synaptic exocytosis enhanced in rat cerebellar granule neurons cultured under survival-promoting conditions.

Cultured rat cerebellar granule neurons (CGNs) are often used to analyze activity-dependent neuronal selection occurring during brain development. The CGNs survive long only when the culture medium contains a depolarizing agent. However, it is argued whether the depolarization critical for survival is of presynaptic or postsynaptic compartment. Since CGNs form no synapses among them, it is generally assumed that the latter would be the case. But it is possible that the depolarization would induce exocytosis of survival-promoting substances whether or not CGNs form synapses. Here we directly examined the exocytotic activities of CGNs under survival-promoting and survival-limiting conditions by electron microscopy to support this possibility. CGNs possessed clusters of synaptic vesicle-like vesicles (SVVs) in neuritic varicosities. CGNs cultured in high-KCl medium had significantly smaller SVV clusters than those cultured in low-KCl medium. The number of SVVs increased when the high KCl-cultured CGNs were transferred to low-KCl medium, indicating a sustained high rate of exocytosis in high-KCl medium. The majority of the varicosities containing SVVs were not apposed to definite postsynaptic structures, indicating that exocytosis occurs from a non-synaptic surface. Fluorescence Ca(2+) imaging revealed that the high KCl-cultured CGNs had spots of high Ca(2+) along their neurites, corresponding to the varicosities.