Increase in number of functional release sites by cyclic AMP-dependent protein kinase in cultured neurons isolated from hippocampal dentate gyrus
Introduction
The regulation of synaptic transmitter release is one mechanism of synaptic plasticity underlying learning and memory (Nayak and Browning, 1999). In vertebrate central neurons, this presynaptic mechanism is likely to be involved in synaptic plasticity in various regions such as CA3 of the hippocampus (Weisskopf and Nicoll, 1995) and the lateral amygdala (McKernan and Shinnick-Gallagher, 1997). Long-term potentiation (LTP) of synaptic transmission between mossy fibers (MFs) and CA3 pyramidal neurons is independent of post-synaptic NMDA receptor activation (Zalutsky and Nicoll, 1990), and both induction and expression mechanisms of LTP are attributable to presynaptic mechanisms (Weisskopf and Nicoll, 1995) involving the activation of the adenylyl cyclase 1 (AC1)-cAMP- protein kinase A (PKA) cascade (Weisskopf et al., 1994, Huang et al., 1994, Huang et al., 1995). The activation of AC in synaptosomes prepared from the hippocampal CA3 region has been shown to enhance glutamate release by increasing the Ca2+-dependent release probability and the size of the readily releasable pool (Lonart et al., 1998). The induction of LTP at autapse in l-AP4-sensitive single-cell cultures of hippocampal dentate gyrus (DG) cells is dependent on Ca2+ influx and PKA activation; this also suggests a presynaptic mechanism involving the activation of previously silent synaptic release sites (Tong et al., 1996). However, no direct evidence supports this interpretation.
Functional synaptic release sites of living neurons can be visualized with styryl fluorescent dyes, FM4-64 and FM1-43. These dyes are trapped within recycling synaptic vesicles and are detected upon release in the peripheral (Betz et al., 1992, Betz and Bewick, 1993) and central (Ryan et al., 1993, Ryan and Smith, 1995, Ryan et al., 1996) nervous systems. These dyes are usually located in synaptic vesicles of cultured neurons by treatment with high-concentration KCl. However, this treatment is too strong for MF synapses. Electrical field stimulation is milder and more physiological, but it is difficult to uniformly activate scattered, presynaptic terminals in a dispersed culture of neurons. The microisland culture of a single DG neuron is useful for the quantitation of functional release sites using FM dyes, because all synaptic terminals originate from a single DG cell, and all synaptic release sites are located on a single cell. Thus, autapses of DG neurons provide a relatively homogeneous synaptic population for image analysis, although there is still some heterogeneity (Murthy et al., 1997). Furthermore, moderate electric field stimulation of a single DG cell in a microisland culture can elicit synaptic exocytosis through an action potential, that is more of a physiological stimulation rather than a direct stimulation of release sites with KCl solution at high concentration.
To obtain evidence that supports the hypothesis that the activation of cAMP-PKA increases the number of synaptic release sites in MF synapses, we determined and compared the number of functional release sites in hippocampal DG neurons before and after activation of the AC-cAMP-PKA cascade. Our results indicate that the activation of the cAMP-PKA cascade increase the number of release sites of DG neuron in a microisland culture.
Section snippets
Cell culture
The microisland method was used to culture hippocampal DG neurons (Yamaguchi et al., 1997). Briefly, rat astrocytes were plated and grown in rat tail collagen spots, which were sprayed on agarose-coated coverglass. The hippocampal dentate gyrus was dissected from 1 to 3-day-old rats. The CA3 region was dissected for culture of CA3 neurons. Neurons were dissociated using papain and cultured on glial microislands in DME (Gibco BRL, New York) medium with sera (8% fetal calf serum, 5% horse serum,
Visualization of functional release sites by loading of dyes FM4–64 and FM1–43
Using fluorescent dyes, FM4–64 and FM1–43, we quantitated the functional release sites in hippocampal DG neurons in a microisland culture in response to activation of the cAMP cascade. To count the number of release sites using FM1–43, synaptic terminals are typically stained and destained by strong depolarizing treatments (reviewed in Cochilla et al., 1999). Since presynaptic LTP of mossy fiber synapses can be induced by trains of depolarizing steps (Zalutsky and Nicoll, 1990), strong
Discussion
Mossy fiber LTP is induced by the activation of the presynaptic cAMP-PKA cascade (Frey et al., 1993, Huang et al., 1994, Weisskopf et al., 1994, Villacres et al., 1998), and the cAMP cascade may facilitate presynaptic exocytosis by increasing the number of release sites in cultured DG neurons (Tong et al., 1996). Using FM dyes, we clearly demonstrated that the activation of the cAMP-PKA cascade increases the number of release sites in single-cell cultures of DG neurons.
DG neurons labeled with a
Acknowledgements
This work was partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Toyota RIKEN and CREST, JST to K.Y. We thank Dr R.T. Kado for critical reading of this manuscript. We also thank Ms K. Kaneko for her excellent technical support.
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