Keiko Tominaga-Yoshino

Repetition of mGluR-dependent LTd causes slowly developing persistent reduction in synaptic strength accompanied by synapse elimination

Synaptic plasticity, the cellular basis of memory, operates in a bidirectional manner. LTP (long-term potentiation) is followed by structural changes that may lead to the formation of new synapses. However, little is known whether LTD (long-term depression) is followed by morphological changes. Here we show that the repetitive induction of metabotropic glutamate receptor (mGluR)-dependent LTD in stable cultures of rat hippocampal slices led to a slowly developing persistent (ranging over weeks) reduction in synaptic strength that was accompanied by the loss of synaptic structures. LTD was induced pharmacologically 1-3 times at 24-h intervals by applying aseptically ACPD (1-aminocyclopentane-1,3-dicarboxylic acid), an agonist of group I/II mGluR, and APV (2-amino-5-phosphonovalerate), an antagonist of the NMDA (N-methyl-D-aspartate) receptor. One ACPD/APV application induced LTD that lasted less than 24 h. After three LTD inductions, however, a gradual attenuation of the fEPSP (field excitatory postsynaptic potential) amplitude and a decrease in the number of pre- and postsynaptic structures were observed. The blockade of LTD by an mGluR antagonist or a protein phosphatase 2B inhibitor abolished the development of the synaptic attenuation. In contrast to our previous finding that the repetitive LTP induction triggered a slowly developing persistent synaptic enhancement, the incremental and decremental forms of synaptic plasticity appeared to occur symmetrically not only on the minutes-hours time order but also on the days-weeks time order.

Repetitive activation of protein kinase A induces slow and persistent potentiation associated with synaptogenesis in cultured hippocampus

Mammalian brain memory is hypothesized to be established through two phases; short-term plasticity, as exemplified by long-term potentiation (LTP) where pre-existing synapses change transmission efficiency, and long-lasting plasticity where new synapses are formed. This hypothesis, however, has not been verified experimentally. Using cultured hippocampal slices, we show that the repeated induction of late-phase LTP by brief applications of forskolin (FK) led to a slowly-developing long-lasting synaptogenesis, as judged from electrophysiological, cytological and ultrastructural indices. These indices include (1) field postsynaptic potential standardized by field action potential, which should represent the number of synapses per neuron; (2) the amounts of synaptic marker proteins; (3) the number of synaptophysin-immunopositive puncta; (4) the number of dendritic spines per length; (5) the density of synaptic ultrastructures; (6) ultrastructures similar to synapse perforation. Increment in these indices occurred approximately 10 days after FK-application and outlasted the following weeks. The increment depended on the times and intervals of FK-application. A biologically inert FK analogue failed to produce the similar effect. An inhibitor for cyclic AMP-dependent protein kinase (PKA) blocked the synaptogenesis. The cultured brain slice repeatedly exposed to FK should serve as a good model system for the analysis of persistent synaptogenesis possibly related to long-term memory in mammalian CNS.

Long-lasting synaptic loss after repeated induction of LTD: independence to the means of LTD induction.

Short‐ and long‐lasting synaptic plasticity is assumed to be the cellular basis of short‐ and long‐lasting memory, respectively. However, the cellular consequences leading to the long‐lasting synaptic plasticity, assumed to include the processes of synapse formation and elimination, remain unknown. Using hippocampal slices maintained stably in culture, we found previously that the repeated induction of long‐term potentiation (LTP) triggered a slowly developing long‐lasting enhancement in synaptic transmission strength accompanied by synapse formation, which was separate from LTP itself. We recently reported a phenomenon apparently of a mirror‐image effect. The repeated activations of metabotropic glutamate receptor (mGluR), which induces long‐term depression (LTD), triggered a long‐lasting reduction in synaptic strength accompanied by synapse elimination. To clarify whether the reported long‐lasting effect was specific to the drugs used previously and whether the effect was specific to mGluR‐mediated LTD, we exposed the cultured slices repeatedly to another Group I metabotropic glutamate receptor (mGluR) agonist, an N‐methyl‐d‐aspartate receptor agonist, and a Na+/K+‐pump inhibitor. All these treatments resulted in an equivalent long‐lasting synaptic reduction/elimination when repeated three times, indicating that the repeated LTD induction leads to synapse elimination. The independence of synapse elimination to the means of LTD induction suggests that the signals leading to short‐term plasticity and long‐term plasticity are independent. Detailed inspections in the representative case of mGluR activation revealed that the reduction in synaptic strength developed with a ∼1‐week delay from the decrease in the number of synaptic structures. This synapse elimination should be unique as it is activity‐dependent rather than inactivity‐dependent.

Persistent synapse loss induced by repetitive LTD in developing rat hippocampal neurons.

Synaptic pruning is a physiological event that eliminates excessive or inappropriate synapses to form proper synaptic connections during development of neurons. Appropriate synaptic pruning is required for normal neural development. However, the mechanism of synaptic pruning is not fully understood. Strength of synaptic activity under competitive circumstances is thought to act as a selective force for synaptic pruning. Long-term depression (LTD) is a synaptic plasticity showing persistent decreased synaptic efficacy, which is accompanied by morphological changes of dendritic spines including transient retraction. Repetitive induction of LTD has been shown to cause persistent loss of synapses in mature neurons. Here, we show that multiple, but not single, induction of LTD caused a persistent reduction in the number of dendritic synapses in cultured rat developing hippocampal neurons. When LTD was induced in 14 days in vitro cultures by application of (RS)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist, and repeated three times with a one day interval, there was a significant decrease in the number of dendritic synapses. This effect continued up to at least two weeks after the triple LTD induction. The persistent reduction in synapse number occurred in the proximal dendrites, but not the distal dendrites, and was prevented by simultaneous application of the group I/II mGluR antagonist (S)-a-methyl-4-carboxyphenylglycine (MCPG). In conclusion, we found that repetitive LTD induction in developing neurons elicits synaptic pruning and contributes to activity-dependent regulation of synapse number in rat hippocampal neurons.

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).

Involvement of the p75NTR signaling pathway in persistent synaptic suppression coupled with synapse elimination following repeated long-term depression induction

Synaptic plasticity, especially structural plasticity, is thought to be a basis for long‐lasting memory. We previously reported that, in rat hippocampus slice cultures, repeated induction of long‐term depression (LTD) by application of a metabotropic glutamate receptor (mGluR) agonist led to slowly developing, long‐lasting synaptic suppression coupled with synapse elimination. We referred to this phenomenon as LOSS (LTD‐repetition‐operated synaptic suppression) to discriminate it from conventional single LTD and proposed it as a model for analyzing structural plasticity. Recently, proneurotrophin‐activated p75NTR signaling has been gaining attention as a possible pathway for the regulation of both neuronal apoptosis and synaptic plasticity. In this study, we examined whether this signaling has a role in the establishment of LOSS. The application of anisomycin indicated that, for LOSS to occur, novel protein synthesis is needed within 6 hr after the induction of mGluR‐dependent LTD, which demonstrates that LOSS is an active process and therefore is not due to withering in response to a shortage of trophic factors. Furthermore, we found that pro‐BDNF (a species of proneurotrophins) is newly synthesized within 6 hr after the induction of LTD. We therefore exogenously applied a cleavage‐resistant form of pro‐BDNF, finding synaptic suppression similar to LOSS. LOSS could be abolished by the application of an antibody that binds to and neutralizes p75NTR following repeated LTD induction. These results suggest involvement of the p75NTR signaling pathway in the long‐lasting decremental form of synaptic plasticity.

Dendritic spine dynamics leading to spine elimination after repeated inductions of LTD

Memory is fixed solidly by repetition. However, the cellular mechanism underlying this repetition-dependent memory consolidation/reconsolidation remains unclear. In our previous study using stable slice cultures of the rodent hippocampus, we found long-lasting synaptic enhancement/suppression coupled with synapse formation/elimination after repeated inductions of chemical LTP/LTD, respectively. We proposed these phenomena as useful model systems for analyzing repetition-dependent memory consolidation. Recently, we analyzed the dynamics of dendritic spines during development of the enhancement, and found that the spines increased in number following characteristic stochastic processes. The current study investigates spine dynamics during the development of the suppression. We found that the rate of spine retraction increased immediately leaving that of spine generation unaltered. Spine elimination occurred independent of the pre-existing spine density on the dendritic segment. In terms of elimination, mushroom-type spines were not necessarily more stable than stubby-type and thin-type spines.

Involvement of TrkB- and p75(NTR)-signaling pathways in two contrasting forms of long-lasting synaptic plasticity.

The repetition of experience is often necessary to establish long-lasting memory. However, the cellular mechanisms underlying this repetition-dependent consolidation of memory remain unclear. We previously observed in organotypic slice cultures of the rodent hippocampus that repeated inductions of long-term potentiation (LTP) led to a slowly developing long-lasting synaptic enhancement coupled with synaptogenesis. We also reported that repeated inductions of long-term depression (LTD) produced a long-lasting synaptic suppression coupled with synapse elimination. We proposed these phenomena as useful in vitro models for analyzing repetition-dependent consolidation. Here, we hypothesized that the enhancement and suppression are mediated by the brain-derived neurotrophic factor (BDNF)-TrkB signaling pathway and the proBDNF-p75NTR pathway, respectively. When we masked the respective pathways, reversals of the enhancement and suppression resulted. These results suggest the alternative activation of the p75NTR pathway by BDNF under TrkB-masking conditions and of the TrkB pathway by proBDNF under p75NTR-masking conditions, thus supporting the aforementioned hypothesis.

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

J. Neurochem. (2012) 122, 81–93.

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 μM, 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.