Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca²⁺ uncaging experiments did not reveal changes in the intracellular Ca²⁺ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca²⁺ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca²⁺ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca²⁺ is explained by the simple mechanism of rapid Ca²⁺ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca²⁺ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca²⁺ channels.

Communication between neurons is largely mediated by chemical synapses. Synaptic strengths are not fixed, but change dynamically in the short and longer term in an activity-dependent manner (short- and long-term plasticity, 1–3). Moreover, neuromodulators act on presynaptic terminals to modulate synaptic strength. Such activity-dependent or modulatory changes are often mediated by the activation of second messengers, such as protein kinase A and C (2). Second messenger systems, particularly the cyclic adenosine monophosphate (cAMP)/PKA-dependent system, are important for higher brain functions, including learning and memory in Aplysia (3), flies (4, 5), and the mammalian brain (6). Despite its functional importance, the cellular and molecular mechanisms of cAMP-dependent modulation are still poorly understood regardless of whether Aplysia synapses and Drosophila neuromuscular junctions have been investigated (2, 7). Mammalian central synapses are no exception here, also reflecting technical difficulties due to the generally small size of the presynaptic terminals in the mammalian brain.Hippocampal mossy fiber-CA3 (MF-CA3) synapses are characterized by exceptionally large presynaptic terminals (hippocampal mossy fiber bouton, hMFB), which allow for the direct analysis of the cellular mechanisms of synaptic transmission and plasticity by using patch-clamp recordings (8–10). Thus, hMFBs provide a suitable model of cortical synapses in the mammalian brain. Moreover, these synapses are functionally important for brain function such as pattern separation (11). Mossy fiber synapses are known to exhibit unique presynaptic forms of short- and long-term synaptic potentiation and depression, which share the cAMP/PKA-dependent induction mechanism (12–15). In addition, the cAMP-dependent plasticity pathway is important for presynaptic modulation by dopamine and noradrenaline (16–18), which modulates hippocampal network activity and behavior. However, its underlying cellular mechanisms remain largely unclear. Enhancement of the molecular priming and docking of synaptic vesicles at mossy fiber synapses has been suggested by previous studies using genetics and electron microscopy (19–21). In particular, RIM1, an active zone scaffold protein, is crucial for cAMP-dependent long-term potentiation (LTP) (19) and is phosphorylated by PKA, although a corresponding phosphorylation mutant of RIM1 was found to have no effect on long-term potentiation (22, but see ref. 23). Other studies on hMFBs have implicated a role in positional priming, i.e., changes in the spatial coupling between Ca²⁺ channels and the release machinery (24). However, there is a lack of the direct visualization or manipulation of this regulation.In order to measure the intracellular Ca²⁺ sensitivity of transmitter release directly and examine the mechanisms of cAMP-dependent modulation quantitatively, we here carried out Ca²⁺ uncaging experiments at hippocampal mossy fiber synapses. Unexpectedly, our results failed to show changes in Ca²⁺ sensitivity, but instead uncovered an increase in local Ca²⁺ concentrations at the release sites. Furthermore, by live imaging of local Ca²⁺ using total internal reflection fluorescence (TIRF) microscopy as well as superresolution time gated STED (gSTED) microscopy, we provided evidence that rather rapid Ca²⁺ channel accumulation may underlie cAMP-induced potentiation instead of release machinery modulations. This study thus provides a potential mechanism of presynaptic modulation at central synapses.