Presynaptic nanodomains: a tale of two synapses

Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release.

Introduction to the Calcium Domain Concept

The concept of local “domains” of intracellular calcium concentration developed from mathematical models of the diffusion of Ca ions (Ca²⁺) away from the mouth of open calcium channels. The first published model, by Chad and Eckert (1984), laid out several key features of this concept. This paper proposed that opening of individual Ca²⁺ channels could form discrete spatial domains where Ca²⁺ concentration is very high locally—within nanometers of a single open Ca²⁺ channel—but drops off steeply with distance away from the channel. In addition, increasingly large depolarizations of the membrane potential should have dual effects: increasing the number of such domains, by increasing the number of open Ca²⁺ channels, yet reducing the magnitude of the local Ca²⁺ concentration change within a domain because of the reduced unitary current through an open channel.

Simon and Llinás (1985) independently developed the Ca²⁺ domain concept and considered the possible role of such domains in triggering of neurotransmitter release from presynaptic terminals. In addition to confirming the deductions of Chad and Eckert (1984) regarding the basic properties of Ca²⁺ domains, they further concluded that these domains usually do not overlap appreciably for the case of squid presynaptic terminals. When many Ca²⁺ channels are opened by large, prolonged depolarizations, there can be a small degree of spatial overlap due to summation of Ca²⁺ from adjacent open channels. The modeling studies of Zucker and Fogelson (1986) reached a similar conclusion. Notably, Simon and Llinás (1985) concluded that an endogenous Ca²⁺ buffer could eliminate summation of Ca²⁺ signals associated with neighboring domains, but should have minimal effect on the peak Ca²⁺ concentration change occurring in the immediate vicinity of an open channel.

At its simplest, diffusion of Ca²⁺ from an open channel can be described by the following equation (Smith and Augustine, 1988):

$\left[{\text{Ca}}^{\text{2+}}\right]\left(x,t\right)=\frac{J_{Ca}}{2\pi D_x}erfc\frac{x}{2\sqrt{Dt}}$

where x is distance from the channel, t is time, J_Ca is the flux of Ca²⁺ through a single open channel, D is the diffusion coefficient for Ca²⁺ and erfc is the complementary error function. This equation predicts that (1) while the channel is open, there will be an inverse relationship between Ca²⁺ concentration and distance from the channel; and (2) Ca²⁺ diffusion time will increase with the square of distance. To aid comprehension of these relationships, we provide “rule of thumb” Ca²⁺ diffusion parameters in Table 1. These parameters are to be taken as order-of-magnitude approximations because their precise values will depend on the value chosen for D, as well as the unitary current flowing through the open Ca²⁺ channel and the properties of cellular Ca²⁺ buffering. More complete mathematical treatments can be found elsewhere, for example in Smith and Augustine (1988), Naraghi and Neher (1997) and Neher (1998), as well as in the pioneering domain papers described above.

TABLE 1

Table 1. “Rule of thumb” relationships for Ca²⁺ diffusion within domains.

As mentioned above, it is possible that Ca²⁺ signals from adjacent open Ca²⁺ channels could overlap under some conditions. The degree of overlap leads to several qualitatively different forms of local Ca²⁺ signaling that are depicted in Figure 1. If there is no spatial overlap between the local Ca²⁺ domains, relative to the position of the relevant Ca²⁺ sensor, then a nanodomain occurs (Figure 1A). This type of signal is called a nanodomain because Ca²⁺ signaling will occur over dimensions of nanometers (Kasai, 1993; Schweizer et al., 1995; Augustine et al., 2003). If there is some degree of spatial overlap between neighboring domains and the Ca²⁺ sensor receives contributions from domains summing over a distance of approximately a micrometer, then this is microdomain-style Ca²⁺ signaling (Figure 1B). Finally, if the sensor receives Ca²⁺ ions diffusing over distances greater than a micrometer, due to summation of microdomains, then a radial gradient style of Ca²⁺ signaling is said to occur (Figure 1C). The differences in the spatial range of these different modes of Ca²⁺ signaling necessarily are associated with differences in the speed of Ca²⁺ triggering of biological processes, as indicated by the t-term in equation (1) and in the right column of Table 1.

FIGURE 1

Figure 1. Three types of local Ca²⁺ signaling. (A) Nanodomains arise from Ca²⁺ (blue) diffusing away from a single open Ca²⁺ channel (red) in the presynaptic plasma membrane (orange). The green spherical structure is a synaptic vesicle and a synaptotagmin-like Ca²⁺ sensor is shown protruding from the surface of the vesicle. (B) Microdomains arise from spatial overlap of local Ca²⁺ signals contributed by multiple open Ca²⁺ channels within a micrometer of each other. (C) Terminal-wide radial gradients of Ca²⁺ arise from long-distance diffusion of Ca²⁺ from channels at the plasma membrane. Derived from illustrations in Schweizer et al. (1995) and Augustine et al. (2003).

From a physical perspective, there is no question that local domains of Ca²⁺ signaling occur in the immediate vicinity of an open Ca²⁺ channel. The question addressed in this review is how relevant such domain signaling is for release of neurotransmitters from presynaptic terminals. We will compare Ca²⁺ signaling at two presynaptic terminals whose large size makes them particularly amenable to experimental analysis: the squid giant synapse and the mammalian calyx of Held. From results obtained at these synapses, as well as selected results from other synapses, we will develop the argument that nanodomain Ca²⁺ signaling is prevalent at presynaptic terminals that rapidly release neurotransmitters, though microdomain signaling also has been established for some types of presynaptic terminals. Radial gradients are likely to be important only for presynaptic terminals that slowly release neuropeptides or for slower modulatory presynaptic actions of Ca²⁺ within fast-acting synapses, for example Ca²⁺-dependent forms of synaptic plasticity.

Evidence for Nanodomains at the Squid Giant Synapse

Virtually all of the early experimental tests of the role of Ca²⁺ domains in triggering neurotransmitter release were performed at the squid giant synapse. As a result, work at this synapse established most of the experimental paradigms that have been used to probe local Ca²⁺ signaling in presynaptic terminals. In this section we will describe some of these key experiments.

The first clues regarding the possible role of presynaptic domain signaling came from analysis of the kinetics of transmitter release. The minimum synaptic delay between a presynaptic Ca²⁺ “tail” current (following a depolarization to a very positive membrane potential) and the resultant postsynaptic response is very brief, on the order of hundreds of microseconds (Llinás et al., 1981; Augustine et al., 1985). Given the “rule of thumb” relationships for local Ca²⁺ signaling described above (Table 1), this indicates that Ca²⁺ must diffuse on the order of 100 nm or less before binding to the Ca²⁺ sensor for neurotransmitter release. This is consistent with a nanodomain type of presynaptic Ca²⁺ signal. Further, Simon and Llinás (1985) indicated that the relationship between presynaptic Ca²⁺ current and postsynaptic response could be explained by a model where domains do not overlap appreciably, i.e., nanodomain signaling.

While these initial observations provided indirect support for the role of nanodomain Ca²⁺ signals in triggering transmitter release at the squid giant synapse, they were open to alternative interpretations. However, several additional experiments have greatly bolstered acceptance of this notion. This experimental evidence can be grouped into three categories.

Manipulations of Domain Size and Number

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.

FIGURE 2

Figure 2. Experimental strategies for probing local Ca²⁺ signaling during neurotransmitter release. (A) At a voltage-clamped squid giant synapse, varying external Ca²⁺ concentration changes presynaptic Ca²⁺ influx by keeping number of Ca²⁺ domains constant, while changing the amount of Ca²⁺ within each domain (insets). Under these conditions, the relationship between presynaptic Ca²⁺ current and amount of transmitter release, assessed by amplitude of the postsynaptic current, is a highly non-linear power function. Both presynaptic and postsynaptic responses are normalized to the maximum values measured at the highest external Ca²⁺ concentration. Data replotted from Augustine and Charlton (1986). (B) Pharmacologically broadening the presynaptic action potential at the squid giant synapse varies Ca²⁺ influx by opening additional Ca²⁺ channels, thereby increasing the number of Ca²⁺ domains (insets). Under these conditions, the relationship between presynaptic Ca²⁺ current and amount of transmitter release is a linear function. Both presynaptic and postsynaptic responses are normalized to the values measured under control conditions (narrowest presynaptic action potential). Data replotted from Augustine et al. (1991).

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).

FIGURE 3

Figure 3. Evaluating local Ca²⁺ signaling during broadening of presynaptic action potentials. (A) Diagram of spatial distribution of Ca²⁺ associated with a single open Ca²⁺ channel. (B) If open Ca²⁺ channels are sufficiently far apart, then Ca²⁺ signals associated with each channel will not overlap, creating a nanodomain arrangement (left). In this case, broader presynaptic action potentials will increase the number of discrete domains, leading to a proportional increase in transmitter release and a linear relationship between Ca²⁺ influx and release (right). (C) If open Ca²⁺ channels are close together, then Ca²⁺ signals associated with each channel will overlap, causing an increase in Ca²⁺ concentration (red bracket) within the local Ca²⁺ signal associated with each channel (left). This creates a microdomain Ca²⁺ signal. In this case, broader presynaptic action potentials will increase the Ca²⁺ concentration within each domain, leading to a supralinear increase in transmitter release due to the cooperative triggering of transmitter release by Ca²⁺. This spatial summation will yield a supralinear relationship between Ca²⁺ influx and release (right) when presynaptic action potentials are broadened. After Augustine et al. (1991).

Measurements of Local Calcium Concentration

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.

Differential Effects of Calcium Chelators

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).

FIGURE 4

Figure 4. Differential effects of EGTA and BAPTA on transmitter release at the squid giant synapse. While presynaptic microinjection of the rapid Ca²⁺ chelator, BAPTA, blocks transmitter release (A), microinjection of the slower chelator, EGTA, has no effect. (B) Each family of traces shows five superimposed presynaptic action potentials (Pre), as well as the postsynaptic responses (Post) that they evoke, during the course of chelator injection. While BAPTA reduces synaptic transmission to the point that postsynaptic responses are subthreshold, EGTA has no effect on postsynaptic response strength. Traces originally from Adler 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.

Evidence for Nanodomains at the Calyx of Held Synapse

The giant calyx of Held synapse has been one of the most prominent preparations developed over the past two decades for analysis of synaptic transmission in the mammalian brain (Forsythe, 1994; Borst et al., 1995). Unfortunately, there is conflicting evidence about whether microdomain or nanodomain Ca²⁺ signaling mediates transmitter release at this synapse (Borst and Sakmann, 1996; Fedchyshyn and Wang, 2005). In this section we will survey this evidence and suggest that the conflict can be resolved by concluding that the spatial range of presynaptic Ca²⁺ signaling changes over the course of maturation of this synapse.

The calyx of Held synapse undergoes tremendous morphological remodeling in the course of its development, with its presynaptic terminal transforming from a spoon- or club-like structure with thin filopodia before the onset of hearing (P11–12) to a highly digitated structure with stalks and swellings containing a total of 500–800 active zones at maturity (>P16; Figure 5). Such structural transformations are thought to be important for supporting release and replenishment of synaptic vesicles (SVs), as well as facilitating clearance of neurotransmitter to alleviate postsynaptic receptor desensitization during high-frequency transmission (Trussell, 1999; von Gersdorff and Borst, 2002). In practice, calyces become less visible in brainstem slices for patch-clamping as myelination progresses during maturation, meaning that most early studies on the calyx of Held synapse focused on immature synapses (e.g., P8–11).

FIGURE 5

Figure 5. Age-dependent morphological remodeling of the calyx of Held. Images of the 3-dimensional structure of P8 (A) and P17 (B) calyces filled with the fluorescent marker biotinylated dextran amine (courtesy of G. Grande). The diameter of each calyx sphere is approximately 15–20 micrometers.

Evidence Against Nanodomain Coupling

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).

Evidence for Nanodomain Coupling

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

$I_{EPSC}\propto {\left[I_{Ca}\right]}^n$

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).

FIGURE 6

Figure 6. Developmental change in presynaptic Ca²⁺ signaling at the calyx of Held synapse. (A,B) Developmental tightening of coupling between Ca²⁺ channels and synaptic vesicles boosts the local [Ca²⁺]_i transient seen by the Ca²⁺ sensor (red traces) and the rate of transmitter release (blue traces) as action potentials (black traces) narrow (from Wang et al., 2008). (C) Transformation from microdomain to nanodomain coupling of Ca²⁺ channels to transmitter release. Note that nanodomains are associated with shorter distance between Ca²⁺ channels and synaptic vesicles (SV) both in the plane of the presynaptic plasma membrane, as well as orthogonal to this membrane. This spatial reorganization of Ca²⁺ signaling is associated with translocation of septins (green) away from the active zone during development.

Evidence for Nanodomain Coupling at Other Synapses

The squid giant synapse and calyx of Held synapse are often considered to be specialized synapses because of their extraordinary size, speed and strength of synaptic transmission. Hence nanodomain coupling could be a feature unique to such high-fidelity, excitatory “relay” synapses. However, this clearly is not the case because a number of synapses have now been shown to release neurotransmitters via nanodomain coupling.

In line with experiments suggesting nanodomain coupling of Ca²⁺ influx to transmitter release at the squid giant synapse and calyx of Held, early studies indicated that Ca²⁺ channels are tightly coupled to Ca²⁺ sensors to enable highly efficient transmission at neuromuscular synapses (Yoshikami et al., 1989; Delaney et al., 1991). In some cases, opening of a single Ca²⁺ channel is sufficient for triggering of fusion events at the active zones of chick ciliary ganglion synapses (Stanley, 1993; Gentile and Stanley, 2005).

Initial studies in mammalian synapses appeared to argue in favor of microdomain coupling (Luebke et al., 1993; Takahashi and Momiyama, 1993; Wheeler et al., 1994; Wu and Saggau, 1994; Mintz et al., 1995). These studies combined Ca²⁺ imaging in presynaptic terminals with patch-clamp recordings from postsynaptic neurons and treatment with subtype-specific Ca²⁺ channel blockers. The results collectively showed that not only are multiple Ca²⁺ channels involved in presynaptic Ca²⁺ signaling but also that different subtypes of Ca²⁺ channels can mediate transmitter release at a synapse, leading to the idea that opening of many adjacent Ca²⁺ channels forms microdomains.

It should be noted that the conditions employed in these different experiments differ in many ways. For example, the fraction of Ca²⁺ channels activated by an action potential differ, ranging from ~10% at the squid giant synapse to >70% at some mammalian central synapses. In addition, extracellular Ca²⁺ concentrations ranged from 11 mM for the squid synapse to 1–2 mM for mammalian central synapses and experimental temperatures varied from 15 to 37^°C. All of these variables profoundly influence the number of Ca²⁺ channels opened by an action potential, as well as the driving force on Ca²⁺ and the kinetics of Ca²⁺ diffusion within nerve terminals, thereby affecting the coupling of Ca²⁺ channels to transmitter release.

Under experimental conditions where these variables have been somewhat controlled and are comparable to conditions employed at the developing calyx of Held synapse, emerging evidence suggests that several conventional mammalian central synapses employ nanodomain Ca²⁺ signaling (Eggermann et al., 2011). For example, nanodomain Ca²⁺ signaling is found both at hippocampal basket cell-granule cell inhibitory synapses and at cerebellar parallel fiber-Purkinje cell excitatory synapses (Bucurenciu et al., 2008; Eggermann and Jonas, 2011; Schmidt et al., 2013). In addition, transmission at sensory synapses other than the calyx of Held apparently employ nanodomain coupling (Brandt et al., 2005; Jarsky et al., 2010). Together, results from a wide variety of mammalian synapses support the notion that nanodomain coupling is highly conserved and is therefore a general scheme for Ca²⁺ triggering of rapid neurotransmitter release.

Conclusions

We have highlighted key evidence from two giant synapses, the squid giant synapse and the calyx of Held synapse, that firmly establish nanodomains as the predominant mode of Ca²⁺ signaling that couples Ca²⁺ influx through presynaptic Ca²⁺ channels to vesicular fusion and synaptic transmission. There is no doubt that nanodomains exist at many synapses, but it is interesting to note that this mode of Ca²⁺ signaling appears to be primarily associated with synapses for fast-spiking neurons, where synaptic transmission is particularly important for preserving temporal precision of information flow (Yang et al., 2014). Nanodomains with tight Ca²⁺ signaling may be particularly suited to trigger highly synchronized fusion events with minimal synaptic delay, temporal jitter, or asynchronous transmitter release. The narrow action potentials typical of fast-spiking neurons are likely to produce brief Ca²⁺ signals with rapid rise and collapse of local [Ca²⁺]. Brief Ca²⁺ transients will also serve to prevent presynaptic Ca²⁺ overload and facilitate Ca²⁺ clearance during prolonged high-frequency transmission. On the other hand, microdomains with loose coupling between Ca²⁺ channels and Ca²⁺ sensors may allow a broader dynamic range for tuning quantal output at synapses where activity-dependent forms of synaptic plasticity play a key role in dynamically regulating synaptic efficacy (Vyleta and Jonas, 2014).

Given the dimensions of SV and active zones, it is reasonable to postulate that the nanodomain Ca²⁺ signaling requires specific molecular specializations to optimize physical interactions between Ca²⁺ channels and SV (Catterall and Few, 2008; Südhof, 2013). To advance our understanding of local Ca²⁺ signaling in presynaptic terminals, it will next be important to define the specific molecular specializations underlying both microdomain and nanodomain Ca²⁺ signaling, and how these two modes of Ca²⁺ signaling are regulated during development. Emerging evidence indicate that several presynaptic proteins help tether Ca²⁺ channels to SV; these proteins include RIM (Kiyonaka et al., 2007; Kaeser et al., 2011), Munc 13 (Chen et al., 2013), bassoon (Davydova et al., 2014) and even synaptotagmin (Young and Neher, 2009). Genetic deletion of the cytometrix protein Bruchpilot loosens the coupling of Ca²⁺ channels and sensors at Drosophila presynaptic terminals (Kittel et al., 2006). In contrast, knockout of septin 5, a filamentous protein that interacts with the SNARE proteins that mediate exocytosis, transforms microdomain coupling typical of immature synapses to nanodomain coupling with functional and morphological phenotypes nearly identical to mature calyces (Figure 6C; Yang et al., 2010). These results suggest that these two modes of local Ca²⁺ signaling are differentiated by specific molecular substrates and potentially could be inter-convertible to confer distinctive Ca²⁺ signaling properties to synapses.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Karen Chung for her help in preparing this paper. This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI 2009-003) (to George J. Augustine), and by Operating Grants from the Canadian Institutes of Health Research (MOP-77610, MOP-81159, MOP-14692, VIH-105441) and Canada Research Chair (to Lu-Yang Wang).

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