Since the classic work of Dodge and Rahamimoff (1967), it has been known that Ca²⁺ acts “cooperatively”, meaning that there is a high-order relationship between extracellular Ca²⁺ concentration and the amount of neurotransmitter release evoked by presynaptic action potentials. Cooperative triggering of transmitter release can be used as a tool to probe the spatial organization of presynaptic Ca²⁺ signaling.

Voltage clamp experiments at the squid giant synapse allowed the first direct measurement of Ca²⁺ entry into a presynaptic terminal and established that this cooperativity arises from a non-linear relationship between Ca²⁺ entry and the resultant release of neurotransmitters. Initially Ca²⁺ entry was varied by depolarizing the presynaptic membrane potential to different levels, thereby varying the number of open Ca²⁺ channels. While an initial account proposed a linear relationship between Ca²⁺ entry and transmitter release (Llinás et al., 1981), a follow-up study that optimized spatial control of presynaptic membrane potential indicated a third- or fourth-power relationship (Augustine et al., 1985). Because depolarizing to different membrane potentials changes the number of Ca²⁺ domains, as well as the Ca²⁺ concentration within each domain, these results are not easily interpreted in terms of local Ca²⁺ signals. A follow-up study by Augustine and Charlton (1986) circumvented this problem by depolarizing to a constant presynaptic membrane potential, to open a constant number of Ca²⁺ channels, while varying Ca²⁺ entry by changing external Ca²⁺ concentration. Under such conditions, an approximate fourth-power relationship between Ca²⁺ entry and transmitter release was observed (Figure 2A). Given that the number of Ca²⁺ domains will be constant under these conditions (Figure 2A insets), this result reveals that cooperativity occurs within the domains.

A strikingly different result is obtained when Ca²⁺ entry is varied by altering the duration of the presynaptic action potential. Blockade of potassium channels prolongs the duration of the presynaptic action potential and thereby increases Ca²⁺ entry into the terminal by increasing the number of open Ca²⁺ channels (Klein and Kandel, 1980). Under such conditions, a linear relationship between action potential duration and postsynaptic response was observed (Augustine, 1990). Although Ca influx was not measured directly, the known gating properties of the squid presynaptic Ca²⁺ channels allowed calculation of the presynaptic Ca²⁺ current and indicated a linear relationship between action potential duration and Ca²⁺ current magnitude (Augustine, 1990). Thus, there is a linear relationship between Ca²⁺ current and transmitter release when Ca²⁺ influx is varied by changing the number of open Ca²⁺ channels (Figure 2B; Augustine et al., 1991).

Given that cooperativity occurs within a domain (Figure 2A), the fact that increasing the number of domains causes a linear increase in transmitter release (Figure 2B) leads to the conclusion that domains do not overlap during a presynaptic action potential. The logic behind this deduction is shown in Figure 3. In brief, if domains do not overlap then transmitter release will increase linearly according to the number of Ca²⁺ channels opened (Figures 3A,B). If domains overlap, then release will increase supralinearly due to summation of Ca²⁺ from neighboring domains, as well as the cooperative action of Ca²⁺ within a domain (Figure 3C). The results of Figure 2B are consistent with the expectations if domains do not overlap (Figure 3B, right). Thus, domains do not overlap during a presynaptic action potential at the squid giant synapse. Similar lines of experimentation have been used to probe the spatial dimensions of Ca²⁺ signaling in other presynaptic terminals (e.g., Wu and Saggau, 1994; Mintz et al., 1995).

If the Ca²⁺ sensor for neurotransmitter release is located within nanometers of an open Ca²⁺ channel, as postulated by a nanodomain coupling scheme, then the local Ca²⁺ concentration experienced by this sensor should be in the range of tens to hundreds of micromolar (Table 1). Two types of experimental paradigms have tested this premise at the squid giant synapse.

The first experiment compared the ability of Ca²⁺ chelators of differing affinities to block transmitter release. For this purpose, analogs of the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) were microinjected into the giant presynaptic terminal. A complex relationship between Ca²⁺ affinity and chelator efficacy was observed: the most potent inhibitor of transmitter release was dibromoBAPTA, a chelator with intermediate Ca²⁺ affinity (4.9 µM), while chelators with lower or higher affinities were less potent (Adler et al., 1991). A numerical model could predict such results only by assuming that the presynaptic Ca²⁺ sensor for transmitter release encounters Ca²⁺ levels on the order of 100 µM or higher (Augustine et al., 1991). This prediction is consistent with the results of experiments employing photolysis of a caged Ca²⁺ chelator; even raising presynaptic Ca²⁺ concentration to 50 µM produced rates of transmitter release lower than those evoked by a presynaptic action potential (Hsu et al., 1996). Together, these results are compatible with the predictions of a nanodomain coupling model.

The second experiment imaged presynaptic Ca²⁺ concentration during transmitter release. For this purpose, Llinás et al. (1992) used a version of the bioluminescent Ca²⁺-binding protein, aequorin, that was engineered to have an especially low affinity for Ca²⁺. This indicator protein, called n-Aequorin-J, only emits light at Ca²⁺ concentrations in the micromolar range or higher, with the amount of light emission increasing steeply as a function of Ca²⁺ concentration. Based on the amount of bioluminescence emitted by n-Aequorin-J injected into the squid giant presynaptic terminal, Llinás et al. (1992) concluded that presynaptic Ca²⁺ concentration reached 200–300 µM during transmitter release. Further, their measurements indicated that the regions of high Ca²⁺ concentration were quite localized, being a fraction of a square micrometer in area. While there are caveats regarding calibration of this indicator, as well as the spatial resolution of the Ca²⁺ imaging method, the results are compatible with the nanodomain coupling model.

In sum, estimates provided by two complementary experimental approaches indicate that Ca²⁺ concentration rises on the order of hundreds of micromolar in the squid giant presynaptic terminal during transmitter release. Such results favor nanodomain coupling of Ca²⁺ channels to transmitter release at this synapse.

The final experimental paradigm—and the one that has proven most useful for probing local Ca²⁺ signaling in presynaptic terminals—is based on comparison of Ca²⁺ chelators with different rates of Ca²⁺ binding (Adler et al., 1991). BAPTA binds Ca²⁺ very rapidly, with its on rate being diffusion-limited, while ethylene glycol tetraacetic acid (EGTA) binds Ca²⁺ more slowly because it is protonated at physiological pH and the bound proton must dissociate before Ca²⁺ can bind. This greatly slows the binding of Ca²⁺ to EGTA in comparison to BAPTA, even though the equilibrium affinity of these two chelators is roughly similar (Augustine et al., 1991). The slow binding of Ca²⁺ allows EGTA to serve as a high-pass temporal (and spatial) filter for Ca²⁺.

Neurotransmitter release evoked by presynaptic action potentials is completely inhibited when BAPTA is microinjected into the squid giant presynaptic terminal (Figure 4A). In contrast, neurotransmitter release is unaffected when EGTA is injected even at concentrations as high as 80 mM (Figure 4B). Injection of EGTA is capable of buffering slower Ca²⁺ signaling processes, such as Ca²⁺ triggering of synaptic augmentation (Swandulla et al., 1991).

The interpretation of these results is that differences in the Ca²⁺ binding rates of these two chelators are responsible for their differential effects on neurotransmitter release. Thus, BAPTA can block transmitter release because it binds Ca²⁺ more rapidly than the Ca²⁺ sensor for transmitter release, while EGTA fails to block transmitter release because it binds Ca²⁺ slower than this sensor. Under conditions appropriate for the squid giant synapse, 80 mM EGTA will bind Ca²⁺ with a time constant of approximately 50 µsec, while the same concentration of BAPTA requires only a fraction of a microsecond (Augustine et al., 1991). Thus, the Ca²⁺ sensor for transmitter release must bind Ca²⁺ in more than 1 µsec and in less than 50 µsec. Knowledge of this temporal constraint has been useful in establishing synaptotagmin as the Ca²⁺ sensor for transmitter release (Hui et al., 2005). In regard to the spatial dimensions of Ca²⁺ signaling during transmitter release, a time window of tens of microseconds means that Ca²⁺ diffuses no more than tens of nanometers prior to triggering transmitter release (Table 1). Thus, a process that is blocked by BAPTA but not by EGTA must be mediated by nanodomain Ca²⁺ signaling and this is the case for transmitter release at the squid giant synapse.

In summary, a variety of different experimental paradigms all point to the conclusion that transmitter release at the squid giant synapse is caused by nanodomain-style coupling of presynaptic Ca²⁺ channels to the presynaptic Ca²⁺ sensor, which presumably is synaptotagmin. In the next section we will see how these and other paradigms have been used to probe the spatial dimensions of presynaptic Ca²⁺ signaling at a different “giant” synapse, the mammalian calyx of Held.

Using recordings of presynaptic Ca²⁺ currents (I_Ca) and excitatory postsynaptic currents (I_EPSC) from young (P8–11) calyx of Held synapses, Borst and Sakmann (1996) first demonstrated that the amplitude of excitatory postsynaptic currents (EPSCs) follows the fourth power of presynaptic Ca²⁺ influx, similar to the squid giant synapse (Figure 2A). However, unlike the squid giant synapse, injection of EGTA at a concentration as low as 1 mM attenuated EPSCs by about 50%, leading to the conclusion that Ca²⁺ influx from many open channels (specifically >60) triggers fusion of a single synaptic vesicle. Furthermore, N-, P/Q, and R-type Ca²⁺ channels were found to jointly mediate transmitter release at this synapse, with P/Q-type Ca²⁺ channels being the most prominent subtype (Forsythe et al., 1998; Iwasaki and Takahashi, 1998; Wu et al., 1998, 1999). By photolysis of caged Ca²⁺ and computer simulations with Ca²⁺ buffer models (Bollmann et al., 2000; Schneggenburger and Neher, 2000), it was estimated that the peak Ca²⁺ concentration at Ca²⁺ sensors on SV is as low as 10 µM, an order of magnitude lower than that deduced for the squid giant synapse. These results are in striking contrast to what would be expected for nanodomain coupling, arguing in favor of a microdomain mode of Ca²⁺ signaling at the immature calyx of Held synapse (Meinrenken et al., 2002).

In parallel with morphological remodeling at the developing calyx of Held, presynaptic action potentials undergo a dramatic shortening in their duration (Taschenberger and von Gersdorff, 2000), which would reduce the number of activated Ca²⁺ channels and thereby attenuate Ca²⁺ influx. Given that the relationship between I_Ca and I_EPSC is a power function (2)IEPSC∝[ICa]n

where n denotes Ca²⁺ cooperativity (typically a value of 3–5; Augustine et al., 1985), spike narrowing would lead to a significant reduction in I_EPSC. In fact, quantal output is increased in older mice (Joshi and Wang, 2002) or maintained in rats (Iwasaki and Takahashi, 2001). This created a paradox: how could the fewer Ca²⁺ channels recruited by narrower action potentials during development somehow become more effective in triggering transmitter release (Yang and Wang, 2006; Kochubey et al., 2009; Leão and von Gersdorff, 2009)?

Two possible mechanisms have been considered: (1) the spatial coupling between open Ca²⁺ channels and Ca²⁺ sensors tightens to yield nanodomain signaling; and/or (2) the Ca²⁺ sensor (e.g., synaptotagmin) becomes more sensitive to Ca²⁺ to detect smaller rises in presynaptic Ca²⁺ concentration. To test the first possibility, Fedchyshyn and Wang (2005) injected EGTA (10 mM) into developing calyx of Held synapses and found that while this slow Ca²⁺ buffer potently attenuates transmitter release in immature terminals (P8–12 mice), as previously reported (Borst and Sakmann, 1996), EGTA had surprisingly little effect at mature synapses (P16–18). This indicates that the distance between Ca²⁺ channels and Ca²⁺ sensors shortens over the course of development. A similar conclusion was reached by using capacitance measurements of exocytosis under different Ca²⁺ buffering conditions (Leão and von Gersdorff, 2009). By using a depolarizing paradigm that recruits an increasing number of Ca²⁺ channels without changing the kinetics of I_Ca or the driving force for Ca²⁺, Fedchyshyn and Wang (2005) further demonstrated that Ca²⁺ cooperativity values (n) were significantly higher in immature synapses than in mature synapses. Based on the logic depicted in Figure 3, this suggests that the number of Ca²⁺ channels required for triggering release of a synaptic vesicle decreases. It has also been shown that while transmitter release at P8–12 synapses requires both N- and P/Q-type Ca²⁺ channels, only P/Q-type Ca²⁺ channels are involved at mature synapses. This suggests that nanodomains contain specific type of Ca²⁺ channels as part of their molecular architecture.

To test the second possibility, Ca²⁺ sensor sensitivity was measured in the developing calyx of Held synapse via simultaneous Ca²⁺ imaging and flash photolysis of caged Ca²⁺ chelators (Wang et al., 2008; Kochubey et al., 2009). Flash photolysis of caged Ca²⁺ has the advantage of bypassing Ca²⁺ entry through Ca²⁺ channels, instead directly triggering release by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a spatially homogeneous manner. Surprisingly, Ca²⁺ sensor sensitivity decreases over development (e.g., K_D values rise from ~80 µM for P9–P11 vs. ~120 µM for P16–P19 synapses) while cooperativity (n) remains the same. Taken together, these studies led to the conclusion that the spatial coupling between Ca²⁺ channels and Ca²⁺ sensors switches from microdomain to nanodomain signaling.

Such changes can account for the upregulation of transmitter release over the course of development of the calyx of Held synapse. Computer simulations with a linearized buffered Ca²⁺ diffusion model and a five-site kinetic model of transmitter release (Naraghi and Neher, 1997; Schneggenburger and Neher, 2000) deduced that during development the peak Ca²⁺ concentration seen by a Ca²⁺ sensor during an action potential increases from 35 to 56 µM (Figures 6A,B). This is a consequence of tightened spatial coupling between Ca²⁺ channels and sensors; the modeling results indicate that the microdomain signaling during an action potential in immature synapses results from 12 Ca²⁺ channels with 50% open probability and 61 nm separation, while the nanodomain coupling at mature synapses results from 9 Ca²⁺ channels with 35% open probability and 23 nm spacing (Figure 6C; Wang et al., 2008, 2009). This change in the spatial dimensions of presynaptic Ca²⁺ signaling nearly doubles the release rate and enables the peak of the local [Ca²⁺]_i transient to occur ~410 µs earlier in mature synapses than in immature synapses (Figures 6A,B). Although direct evidence for the topographic organization of Ca²⁺ channels at the developing calyx remains elusive, these simulation results provide a theoretical framework for envisaging how a developmental transition from microdomain to nanodomain coupling can recapitulate a number of experimental observations, including an explanation for how narrower action potentials become more effective in triggering transmitter release (Taschenberger and von Gersdorff, 2000).