Elsevier

Brain Research

Volume 1042, Issue 1, 25 April 2005, Pages 6-16
Brain Research

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

https://doi.org/10.1016/j.brainres.2005.01.102Get rights and content

Abstract

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).

Introduction

Long-lasting plasticity, the cellular basis for memory lasting for periods longer than days–weeks, is thought to be substantialized by the formation of new synapses, which occur consecutively to short-lasting plasticity, the rapidly occurring augmentation of synaptic strength in existing synapses [2]. However, the process of conversion from short-lasting plasticity into the long-lasting one remains poorly understood, mainly because of the lack of a good model that allows for the prolonged examination of synapse formation. Several recent reports have revealed that morphological changes take place in the synaptic structures accompanying long-term potentiation (LTP) in the hippocampus [7], [17], [28]. Expecting those morphological changes to represent the conversion process, an increasing number of researchers have begun analyses of the cellular and molecular mechanisms of those morphological changes [19], [33]. However, it remains unknown whether those morphological changes, the first indications of which are recognized within minutes–hours order of time after LTP induction, truly represent the conversion process that leads to the formation of synapses, which are preserved for days–weeks. This question arises from the viewpoint that if all LTPs were to proceed to synaptogenesis lasting for days–weeks, it would be difficult to assume a process of selection by which information to be stored is segregated from that to be erased. In other words, if the morphological changes accompanying LTP were truly the conversion process, the selection would have taken place before, not after, the occurrence of LTP. It is rather conceivable, however, that the selection would take place after the occurrence of LTP, which would represent the transient storage of information in the existing synapses.

Using stable cultures of hippocampal slices, we have shown recently [26], [27] that repeated induction of LTP by repeated activation of PKA (cyclic-AMP-dependent protein kinase) led to long-lasting enhancement of excitatory postsynaptic potential (EPSP). This synaptic enhancement that lasted more than 3 weeks was accompanied by an increase in the number of morphologically identifiable synaptic structures, but not by an increase in the number of neurons. It should be pointed out that a single induction of LTP produced only short-lasting (i.e., lasting less than 24 h) synaptic enhancement that could not be converted into a long-lasting (i.e., lasting for days) one. However, the repetition of LTP induction to produce this enhancement must be spaced by appropriate intervals. To discriminate this repetitive LTP-induced synaptic enhancement from the conventional single LTP, we call this phenomenon “RISE” in this report. The above findings suggest that the selection process would lie in the repetition of LTP and that the morphological changes following not the single induction but the repetitive LTP are involved in the conversion from the short-lasting plasticity into the long-lasting one.

The RISE in cultured brain slices may serve as a model phenomenon for the analyses of the cellular mechanisms underlying the conversion process. If an equivalent phenomenon was reproduced in the dissociated neuronal cell culture, where original neural circuits are once disorganized and ectopically rebuilt, it would support the view that the repetitive activity-dependent synaptogenesis is an intrinsic property of neurons. In addition, it would provide us a wider array of tools for the analyses of the conversion process, as the dissociated cell culture system has better accessibility to morphological and pharmacological examinations than slice culture system (in spite of disadvantages including loss of neural circuits in vivo). The results are promising.

Section snippets

Cell culture

Dissociated hippocampal neuronal cultures were prepared according to conventional methodology [22]. Briefly, the hippocampi were isolated from Wistar/ST rat (supplied by Nihon SLC) embryos of gestational day 18 and the cells were dispersed by trypsination and trituration. The cells were plated on polyethyleneimine-coated glass coverslips at the density of 1 × 104 per cm2. We adopted this relatively low density of neurons to facilitate identification of the origin of the neurite under

Short- and long-lasting increase in synapse number

In this study, we identified synapses by immunostaining with pre- and postsynaptic markers. Not all of the Syp-immunopositive puncta were synapses, and not all of the Drb-immunopositive puncta were synapses, either. Axon terminals and dendritic spines lacking their counterparts were present. Hence, we stained the cultures with the two markers (Fig. 2) and identified a synapse as an apposition site of a Syn-immunopositive punctum and a Drb-immunopositive punctum. The functionality of those sites

Necessity of a model for cell-biological analysis of long-lasting plasticity

It is assumed that short-lasting plasticity occurring in existing synapses is converted into a long-lasting one through the formation of new synapses. The mechanism of conversion remains unclear, however, mainly because of the lack of a good model for analysis. The required model should be an in vitro one to which prolonged experimental manipulations (including physiological, pharmacological, biochemical, and genetic ones) are applicable and with which ongoing morphological changes are

Acknowledgments

We thank Y. Kuroda, Y. Shinoda, N. Taniguchi, and T. Tashiro for discussion and encouragement. This study was supported by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology to K. T.-Y. and from CREST to Y. Kuroda.

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