Regulation of density of functional presynaptic terminals by local energy supply.

BACKGROUND
The density of functional synapses is an important parameter in determining the efficacy of synaptic transmission. However, how functional presynaptic terminal density is regulated under natural physiological conditions is still poorly understood.


RESULTS
We studied the factors controlling the density of presynaptic functional terminals at single dendritic branches of hippocampal neurons and found that elevation of intracellular Mg(2+) concentration was effective in increasing the density of functional terminals. Interestingly, the upregulation was not due to synaptogenesis, but to the conversion of a considerable proportion of presynaptic terminals from nonfunctional to functional. Mechanistic studies revealed that the nonfunctional terminals had inadequate Ca(2+)-sensitivity-related proteins, resulting in very low Ca(2+) sensitivity within their vesicle release machinery. We identified energy-dependent axonal transport as a primary factor controlling the amount of Ca(2+)-sensitivity-related proteins in terminals. The elevation of intracellular Mg(2+) enhanced local energy supply and promoted the increase of Ca(2+)-sensitivity-related proteins in terminals, leading to increased functional terminal density.


CONCLUSIONS
Our study suggests that local energy supply plays a critical role in controlling the density of functional presynaptic terminals, demonstrating the link between energy supply and efficacy of synaptic transmission.

Generally, synaptic silence can be divided into two categories based on the locus of silence, either pre-or postsynaptic. Molecular mechanisms of postsynaptic silence have been studied extensively, determining that lack of AMPA receptors at the postsynaptic locus is largely responsible for postsynaptic silence [13,14,5,15,2]. However, the mechanisms of presynaptic silence are still elusive [16,17].
The presence of nonfunctional presynaptic release sites was first theorized from early quantal analysis studies, and later directly observed with the application of FM dyes, showing a considerable number of nonfunctional presynaptic terminals (with extremely low release probability, Pr) [18][19][20][21]. A large amount of research has been done to understand the mechanisms underlying the presence of nonfunctional terminals. Convergence of evidence suggests that presynaptic silence can be regulated by perturbations in network excitability. For instance, presynaptic terminals can be silenced by chronic depolarization [22,23] or glutamate excitotoxicity [9]. Or, nonfunctional terminals can be activated by chronic (in several hours) blockage of action potentials [23,24], but surprisingly the activated terminals will be silenced again if the blockage is prolonged (>48 hr) [25].
While there is a plethora of information regarding the mechanisms involved in presynaptic nonfunctionalfunctional switching, there are still major unsolved issues.
(1) Is the presynaptic nonfunction (silence) a normal physiological state of presynaptic terminals [16]? If so, what are the physiological parameters that determine the ratio of functional/nonfunctional terminals, and in turn, determine the functional synapse density at dendrites? Most of the previous studies used extreme treatments, such as high K + , high concentration of ambient glutamate, intense AP stimulation for hours and complete inhibition of APs for days [9,[22][23][24][25]42]. Studies using these treatments can help understand the regulation of nonfunctional-functional conversion under pathological processes, but still might not provide accurate information on regulation of nonfunctional terminals under natural physiological conditions. (2) What molecular factors are necessary in presynaptic terminals to make them functional under physiological conditions? One strategy to answer this question is to delete presynaptic proteins and look at the loss of presynaptic functions [44]. However, we are more interested in what presynaptic proteins are "missing" in natural state. If the presence of nonfunctional presynaptic terminals is an integral part of physiological regulation of synaptic strength, then identification of the "missing" proteins in presynaptic nonfunctional terminals will help elucidate the molecular mechanisms controlling the presynaptic nonfunctional-functional switching under physiological conditions. Furthermore, modifying the quantity of the identified proteins might serve as a way to regulate the density of functional synapses.
In the current study, we applied bursting action potentials (correlated activity) as input and quantified the functional status of presynaptic terminals in cultured hippocampal neuronal networks by their ability to undergo vesicle turnover. We found in this study that elevating extracellular Mg 2+ concentration results in an increase in intracellular Mg 2+ and subsequently a conversion of a majority of the nonfunctional terminals to functional status. Therefore, we manipulated extracellular Mg 2+ as a tool to help determine the mechanisms involved in this process, including presynaptic Ca 2+ sensitivity, Ca 2+ -sensitivity-related protein turnover and energy supply. In conclusion, our study suggests that local energy supply is an important physiological factor that regulates the ratio of functional/nonfunctional terminals, achieving a regulation of the density of functional terminals. Moreover the concentration of intracellular Mg 2+ might serve as a messenger for such regulation of functional terminal density.

Functional terminal density is determined by intracellular Mg 2+
In the current study, we used bursting action potentials (APs) (correlated activity) as input to quantify the functional synapse density. Bursting APs were chosen because they play an essential role in information transmission in both the developmental and mature networks in the hippocampus [46][47][48][49]. Because bursts in the hippocampus usually contain 2-6 high-frequency (>100Hz) APs under physiological conditions [50], and a special form of bursts, the "theta bursts" (usually 4-5 APs in a burst), is found effective at inducing long-term plasticity [51,52], here we chose 5AP bursts as input stimuli for quantification of the density of functional terminals. The stimuli sequence contained 6 groups of 5AP bursts with an inter-burst-interval of 10 s (30 APs total) (Fig. 1a). FM1-43 or FM4-64 was used to detect vesicle turnover of functional terminals (Fig. 1a) [24]. Here, a terminal was defined as functional if releasable FM dye was detectable following bursting input stimulation. A terminal was considered to be nonfunctional if it failed to release even one vesicle after 30 [53], was administered at 1 μM for 4 hr. Following 4 hr IM administration, N 5AP was increased in concert with the elevation of [Mg 2+ ] i , and the enhancement persisted for~32 hr ( Fig. 2c and f). Based on these data, we concluded that [Mg 2+ ] i might be an important factor in the regulation of functional terminal density.   (Fig. 2d), we used [Mg 2+ ] o elevation in neuronal cultures as a tool to determine the molecular substrates involved in N 5AP regulation.
To start, we examined whether the increase in N 5AP was caused by synaptogenesis. By immunofluorescence (IF), we labeled structural terminals with antibodies against several presynaptic proteins, including synaptophysin (SYP, vesicle protein), VGLUT1 (excitatory vesicle protein), VGAT (inhibitory vesicle protein) and Bassoon (active zone protein) (Fig. 3a). There were no significant changes in the density of presynaptic protein puncta after elevating [Mg 2+ ] o for 4 hr (Fig. 3b), suggesting that elevation of [Mg 2+ ] i might not, at least within 4 hr, induce synaptogenesis.
Notably, at the low [Mg 2+ ] i condition, the 5AP bursting input-induced FM(+) puncta density was remarkably lower (0.73 μm −2 , Fig. 2d control) than the 600APinduced FM(+) puncta density (1.38 μm −2 ). It is possible that this difference was due to the ability of terminals to release vesicles under maximal stimulation but not under 5AP bursting input. To test this possibility, we compared the vesicle release of terminals under 5AP and 600AP stimulations at the same dendrite (Fig. 3c). Indeed, only~50 % of terminals were activated under 5AP stimulation at the low [Mg 2+ ] o condition ( Fig. 3c  The quantity of Ca 2+ -sensitivity-related proteins in presynaptic terminals determines the terminal functionality under physiological conditions To further understand how [Mg 2+ ] i affects presynaptic Ca 2+ sensitivity, we investigated the potential molecular mechanisms underlying this phenomenon. Generally, Ca 2+ sensitivity of presynaptic terminals is determined by three biophysical factors (illustrated in Fig. 4a): (1) the quantity of presynaptic Ca 2+ channels [56] (mainly Ca v 2.1 and Ca v 2.2 in hippocampal terminals [57]), which conduct the Ca 2+ influx; (2) the quantity of Ca 2+ sensor proteins (mainly Synaptotagmin1 [SYT1] for excitatory central synapses [58]), which couple the Ca 2+ signals to vesicle release; and (3) the distance from Ca 2+ channels to vesicle release machinery, which determines the coupling efficacy of the Ca 2+ influx to the operation of release machinery. The coupling efficacy is regulated by multiple vesicle and active zone proteins (e.g. Rab3, RIM1, Munc13, ELKS, Syntaxin1) [44].
To address these three possibilities, we compared the profiles of protein expression in terminals at the low (Ctrl 0.8) versus the high [Mg 2+ ] o condition (1.2 4hr) using IF staining. There was no difference in the expression level of Ca v 2.1 and Ca v 2.2 per area of dendrites ( Fig. 4b and d), eliminating the first possibility. However, the expression of Ca 2+ sensor protein (SYT1) and coupling proteins (e.g. Rab3a, RIM1, Munc13-1, ELKS, Syntaxin1) were significantly higher in terminals at the high [Mg 2+ ] o condition (30-44 %, p < 0.001) ( Fig. 4c and d). These data, together with the observation that the expression of structural (vesicle and active zone) presynaptic proteins (SYP, Bassoon, VGLUT1 and Synapsin1) did not change (Figs. 3a and b, 4b) suggest that elevation of [Mg 2+ ] i increased presynaptic Ca 2+ sensitivity via enhancing the expression of presynaptic proteins critical for controlling presynaptic Ca 2+ efficacy (we refer to these proteins hereafter as presynaptic Ca 2+ -sensitivity-related proteins). 2+ ] i in controlling the efficiency of axonal transport of Ca 2+ -sensitivity-related proteins within several hours

Role of [Mg
We carried out further experiments to explore how elevating [Mg 2+ ] i promoted the augmentation of Ca 2+ -sensitivity-related proteins in terminals. Generally, the quantity of proteins in terminals depends on the three major processes involved in the protein life cycle: (1) synthesis, (2) transport, and (3) degradation, so we investigated if [Mg 2+ ] i affected any of these processes in regard to Ca 2+ -sensitivity-related proteins in terminals.
First, we compared the quantity of SYP and Ca 2+ -sensitivity-related proteins from total proteins extracted from entire neurons at the low (Ctrl 0.8) and high [Mg 2+ ] o (1.2 6 hr) conditions by Western blot. Surprisingly, the quantity of each protein at the high [Mg 2+ ] o condition was not significantly higher (at least within 6 hr) than that at the low [Mg 2+ ] o condition (Fig. 5a), ruling out the possibility that elevating [Mg 2+ ] i could promote the detectable enhancement of synthesis of Ca 2+ -sensitivity-related proteins rapidly (within 6 hr).
Next, we checked whether elevating [Mg 2+ ] i could promote the transport of Ca 2+ -sensitivity-related proteins from soma to axonal terminals. Interestingly immunoreactive fluorescence for the Ca 2+ -sensitivity-related protein  Fig. 4d). These data highly suggested that elevation of [Mg 2+ ] i might accelerate the transport of Ca 2+ -sensitivityrelated proteins from soma to terminals selectively. To study this phenomenon quantitatively, we examined the changes of the quantity of SYT1 in the somatic area and terminals at dendrites by IF after elevating [Mg 2+ ] o from 0.6 to 0.75-1.2 mM in gradient for 4 hr (thereby [Mg 2+ ] i could be clamped at different levels). The immunoreactivity of SYT1 in the somatic area (Fig. 5d, upper) and terminals at dendrites changed in opposite directions, linearly proportional to the Mg 2+ level (Fig. 5d, linear regression).
Third, we examined the effects of elevating [Mg 2+ ] i on the rate of protein degradation in terminals. Assuming that presynaptic terminals do not have the capacity to synthesize new Ca 2+ -sensitivity-related proteins (no evidence has been found that they do), the quantity of presynaptic Ca 2+ -sensitivity-related proteins at equilibrium would be largely determined by the balance of protein transport and degradation. Therefore, if we blocked axonal transport, the rate of protein decline would reflect the rate of protein degradation. When axonal transport was blocked by 0.5 mM Colchicine (Colch) [59], the concentration of the presynaptic proteins studied decreased linearly, as a function of the amount of blocking time ( Fig. 5e and f ), albeit at varying rates. The rate of protein degradation (half-life) for the Ca 2+ -sensitivity-related proteins (e.g. Rab3a, RIM1 and Munc13-1) (several hours) was much faster than that for structure-protein SYP (several days). Interestingly, the elevation of [Mg 2+ ] i did not alter the degradation rate of presynaptic proteins, indicating that degradation rate was not part of the mechanism by which increased [Mg 2+ ] i altered presynaptic Ca 2+ sensitivity ( Fig. 5e and f ).
Altogether, these data suggested that the Ca 2+ -sensitivity-related proteins had a relatively fast degradation rate and thereby their quantities in terminals at dendrites were strongly dependent upon the efficiency of axonal transport. The elevation of [Mg 2+ ] i likely promoted the augmentation of Ca 2+ -sensitivity-related proteins in terminals by increasing the efficiency of their transport from soma to terminals, which ensured sufficient Ca 2+ sensitivity for AP-dependent vesicle turnover (illustrated in Fig. 5i).
Based on these data, we inferred that impairment of axonal transport would reduce the density of functional terminals immediately (within hours). To validate this, we measured the temporal changes of N 5AP after blockade of axonal transport by Colch. Colch treatment did not prevent elevation of [Mg 2+ ] i after increasing [Mg 2+ ] o but it prevented the increase in N 5AP (Fig. 5g). Furthermore, blocking axonal transport at the high [Mg 2+ ] o condition caused a remarkable reduction of N 5AP , without change of [Mg 2+ ] i (Fig. 5h). These data demonstrate the critical role of ongoing axonal transport in maintaining functional status of terminals.

Local energy supply is enhanced by elevation of intracellular Mg 2+
The above studies suggest that the level of intracellular Mg 2+ may determine the efficiency of protein transport in axons. Previous studies indicate that [Mg 2+ ] i and intracellular ATP concentration ([ATP] i ) are two endogenous factors critical for controlling the speed of axonal transport [60]. Therefore, the elevation of [Mg 2+ ] i might improve the efficiency of axonal transport by affecting the efficacy of motor proteins [60], and/or indirectly by promoting energy supply, as Mg 2+ is well known to be necessary for normal mitochondrial function.
We studied the effects of elevating [Mg 2+ ] i on the spatial distribution and function of mitochondria. The function of each mitochondrion was quantified by measuring its membrane potential (ΔΨ), using the ΔΨ-sensitive fluorescent dye JC-1 [61]. ΔΨ increased by~39 % after elevating Mg 2+ for 4 hr and this increase persisted up to several weeks ( Fig. 6a and b). The number of mitochondria per unit area of distal dendritic branches (N mito ) also increased, by~20 % (Fig. 6b). Since N mito and ΔΨ are two key parameters for the determination of mitochondrial function, we used the product of N mito and ΔΨ to represent the total mitochondrial function per unit area of dendritic branches in the synaptic network, and found N mito × ΔΨ increased by~68 % after elevating [Mg 2+ ] o for 4 hr (Fig. 6b) (Fig. 6d).
These data suggest that the elevation of [Mg 2+ ] i increased energy supply in local area of the network.
Local energy supply, Ca 2+ -sensitivity-related proteins and functional terminal density We investigated whether the increased quantity of Ca 2+ -sensitivity-related proteins at terminals and subsequently the functionality of terminals as a result of elevated [Mg 2+ ] i was due to enhanced mitochondrial function and local energy supply at dendritic branches. To address this, we examined whether the quantity of Ca 2+ -sensitivity-related proteins or functional terminal density was correlated with mitochondrial density since both are upregulated after the elevation of [Mg 2+ ] i (Figs. 4d, 6b and d). Mitochondria were stained with the fluorescent marker MitoView633. Then, at the same region, functional terminals responding to 5AP bursts were labeled by FM1-43. Subsequently, structure-and Ca 2+ -sensitivity-related proteins in terminals were stained by IF at the low and high [Mg 2+ ] o conditions (Fig. 7a). We observed that the nonfunctional terminals (the SYP positive but FM1-43 negative puncta) usually lacked some of the Ca 2+ -sensitivity-related proteins, but the type of proteins whose expression was low varied in different nonfunctional terminals. For example, in terminal 1 of Fig. 7a, the expression of SYT1 and ELKS was relatively low, while in terminal 2, the expression of Rab3a, RIM1 and Munc13-1 was relatively low (Fig. 7a white circles: terminal 1, 2). This observation suggested that it was the inadequate expression of multiple Ca 2+ -sensitivity-related proteins, but not specific one of them that made a presynaptic terminal nonfunctional. Therefore, we used ΣQ proteins to represent the total quantity of Ca 2+ -sensitivityrelated proteins (see Methods).
When we compared the relationship of N mito with the quantity of the structure-related protein SYP (Q SYP ) and with ΣQ proteins , we found that ΣQ proteins was correlated with N mito , while Q SYP was not (Fig. 7b lower). Elevating [Mg 2+ ] o increased both ΣQ proteins and N mito (Fig. 7b-d).
The correlation between ΣQ proteins , the ratio of ΣQ proteins /Q SYP and N mito remained (Fig. 7b upper, c and d). Most importantly, N 5AP at the same local area was also linearly correlated with N mito (Fig. 7c and d). Therefore, the close linear correlations between ΣQ proteins , ΣQ proteins / Q SYP , N 5AP and N mito support the notion that local energy supply provided by mitochondria might be important for maintaining high levels of Ca 2+ -sensitivity-related proteins in terminals, which in turn determine the density of functional terminals.
To demonstrate the causal relationship between energy supply and N 5AP , we tested whether modification of energy supply would affect both ΣQ proteins and N 5AP concurrently. Energy supply was increased by elevating  ΣQ proteins and N 5AP . The increase or decrease in [ATP] i was linearly correlated with the increase or decrease in ΣQ proteins and N 5AP (Fig. 7e and f ).
Taking all the data together, we conclude that local energy supply is one of major factors that determine functional terminal density.

Correlation between quantity of Ca 2+ -sensitivity-related proteins and mitochondrial density in intact animals
Finally, we determined whether the in vitro findings could be verified in intact animals. We treated mature male Sprague-Dawley rats (16 months old) with Magnesium L-Threonate (MgT) in drinking water. This treatment is known to be effective in elevating [Mg 2+ ] CSF [62]. After 8 months of MgT supplement (24 months old), we sacrificed the animals, dissected the Hippocampus CA1 Stratum Radiatum region of the brain, and conducted both electron microscopy (EM) and IF.
First we checked whether MgT supplementation could modify mitochondrial density (N mito ). We measured N mito (number of mitochondria per unit area of image) in EM images of each animal (Fig. 8a). Mean N mito in MgT group (0.47 ± 0.02 μm −2 , N = 11 rats) was~26 % higher than that in control group (0.37 ± 0.02 μm −2 , N =10 rats) (Kolmogorov-Smirnov test, p < 0.01) (Fig. 8b). The extent of the increase in N mito by MgT supplementation in intact animals was similar to that in cultured neurons (Fig. 7b).
Next, we checked the expression of structure-related (e.g. SYP) and Ca 2+ -sensitivity-related proteins (e.g. SYT1, Rab3a, RIM1, Munc13-1, ELKS and Syntaxin1) in 70 nm ultrathin slices by IF. The total fluorescent intensity of immunoreactive puncta of Ca 2+ -sensitivity-related proteins was higher in MgT group than that in control group (Fig. 8c). We calculated the quantity of each protein (Q) in each individual rat, and found no correlation between Q SYP and N mito for each rat in both groups (Fig. 8d  lower). However, ΣQ proteins (reactive Ca 2+ -sensitivity-related The experiments were performed using sister cultures for each treatment. The mean ± SEM of coverslips was presented. Linear regression for (b-f) proteins) was linearly correlated with N mito in both groups, and MgT treatment positively shifted the correlation curve (Fig. 8d upper). Thus, the data from intact rats were in agreement with the in vitro data (Fig. 7b). On average, ΣQ proteins was~25 % higher in MgT group (Kolmogorov-Smirnov test, p < 0.0005) than that in control group, suggesting that the administration of MgT can promote the presence of Ca 2+ -sensitivity-related proteins in the Stratum Radiatum layer of CA1.
Furthermore, we checked the quantity of Ca 2+ -sensitivity-related proteins relative to the structure-related protein SYP (i.e. ΣQ proteins /Q SYP ), and found that it was linearly correlated with N mito in both control and MgT groups (Fig. 8e). After MgT supplementation, both N mito and ΣQ proteins /Q SYP increased, maintaining the linear correlation between each other (Fig. 8e). These data were also similar to the in vitro data (Fig. 7c and d).
Altogether these in vivo results were in agreement with the in vitro findings, and further supported our hypothesis that Mg 2+ plays an important role in controlling mitochondrial density in branches, which in turn determines the expression of Ca 2+ -sensitivity-related proteins in terminals.

Presynaptic Ca 2+ sensitivity, intracellular Mg 2+ and functional terminal density
In the current study, we observed that only half of presynaptic terminals were responsive to physiological pattern of input (Fig. 3a-d), which is similar to what is reported in prior studies [21,20,24,63]. We found that elevating [Mg 2+ ] o is very effective in converting these nonfunctional terminals into functional ones (Fig. 2). Increasing [Mg 2+ ] o was used as a tool to elevate [Mg 2+ ] i , resulting in an increase in the ratio of functional/nonfunctional terminals. While elevating [Mg 2+ ] o could lead to the increase of participation of the functional terminals during the enhancement of presynaptic plasticity via boosted NR2B-containing NMDAR-induced retrograde signaling (see our previous study [24]), here we show evidence suggesting that the change in functional terminal density is primarily due to [Mg 2+ ] i . First, [Mg 2+ ] o had a bell-shape relationship with functional terminal density (Fig. 1c) and [Mg 2+ ] i (Fig. 1d), whereas [Mg 2+ ] o is positively correlated with NMDAR upregulation (see Fig. 7 of [24]). Also, [Mg 2+ ] i was positively correlated with functional terminal density (Fig. 2b). Second, imipramine, which can elevate [Mg 2+ ] i via the blockage of Mg 2+ extrusion, temporally altered functional terminal density (Fig. 2c and f ) ] i , we revealed that very low presynaptic Ca 2+ sensitivity was largely responsible for the presence of nonfunctional terminals ( Fig. 3e and f). This low Ca 2+ sensitivity of synaptic release machinery was caused by the insufficient quantity of terminal Ca 2+ -sensitivity-related proteins (e.g. SYT1, Rab3, RIM1, Munc13, ELKS and Syntaxin1) ( Fig. 4c and d). Previous studies suggest the possible association between the lack of presynaptic cytomatrix proteins and presynaptic silencing [41,42,25,43], and have extensively investigated the effects of Ca 2+ -sensitivity-related proteins (as well as their interactions) on transmitter release (see review [44,64]) and on the underlying molecular pathways involved in the regulation of presynaptic release [16,17,26]. However, it remains elusive how to upregulate the expression of Ca 2+ -sensitivity-related proteins in terminals under physiological conditions. The current study showed that elevating [Mg 2+ ] i resulted in more functional terminals within a branch. Increased [Mg 2+ ] i resulted in the conversion of nonfunctional terminals into functional ones (Fig. 2c, d and f), and was associated with an increase in Ca 2+ -sensitivity-related proteins in terminals ( Fig. 4c and d).
These data indicate that intracellular Mg 2+ might be a critical endogenous enhancer of terminal Ca 2+ -sensitivity-related protein quantity, and in turn, a regulator of functional terminal density. Hence, regulating [Mg 2+ ] i may be a valid way to control functional terminal density under physiological conditions. It should be noted that a limitation of our protein turnover analysis experiments (Fig. 5b-f ) was that they did not provide direct evidence that elevated [Mg 2+ ] i accelerated the axonal transport of Ca 2+ -sensitivity-related proteins from soma to terminals, and such experiment should be done in future studies.

Local energy supply, Mg 2+ and functional terminal density
Of all organs or tissues, the brain consumes the highest energy per unit weight [65,66]. Most of the brain's energy consumption goes into sustaining neural activity within networks, with~80 % or more consumed by synaptic transmission (including action potentials and postsynaptic effects) [65,66]. Therefore, the synaptic network needs to maximize the efficiency of information transmission per unit energy expended. This maximization is considered a basic principle in the design of the neural network [67]. To achieve proper neural network functionality, the level of neural activity needs to be scaled to the available energy supply. The results from the current study suggest a possible solution: let local energy supply (E) control the fraction of synaptic terminals that can participate in synaptic transmission (i.e. N ∝ E) (Fig. 7).
In this study (illustrated by the schematic cartoon in Fig. 9), we show that functionality of presyanptic terminals is likely determined by the amount of Ca 2+ -sensitivityrelated proteins in terminals. Interestingly, the half-life of Ca 2+ -sensitivity-related proteins (several hours) was shown to be much shorter than that of structure-related proteins (e.g. SYP) (several days) ( Fig. 5e and f), indicating that their presence at the terminal is a function of transport efficiency. As protein transport in the axon is a high energy-consuming process, the status of local energy supply should directly affect the efficiency of protein transport [60]. When local energy supply is adequate, sufficient amount of Ca 2+ -sensitivity-related proteins will be transported to terminals (Fig. 7a), ensuring that most of terminals are functional in response to physiological inputs ( Fig. 3c and d). However, when energy supply and subsequent protein transport efficiency are reduced, the amount of Ca 2+ -sensitivity-related proteins in terminals will drop significantly (Fig. 7a), resulting in the decline of Ca 2+ sensitivity ( Fig. 3e and f) and consequently the ability of the terminals to participate in synaptic transmission (Fig. 7). Within this construct, it is likely that over-consumption of energy will lead to down-regulation of axonal transport of Ca 2+ -sensitivity-related proteins into terminals, resulting in reduced terminal Ca 2+ sensitivity, in order to save energy. This negative feedback control will ensure that the proportion of terminals in the functional state will always be scaled according to local energy supply (Fig. 9). The possible computational consequence of such an arrangement is that a presynaptic terminal fluctuates its ability to transfer information dependent on the extent of local energy supply: the higher the energy supply, the larger the fraction of presynaptic terminals in the functional state and the stronger the synaptic transmission during information transmission.
We showed that elevating [Mg 2+ ] o can concurrently enhance the number and function of mitochondria in distal branches, resulting in the increase of reserved [ATP] i (Fig. 6c). Currently, we have not determined exactly how the enhancement was achieved after elevation of [Mg 2+ ] o , but a possible mechanism is illustrated in Fig. 9. For the increase in ΔΨ, one possible explanation is that elevating both extra-and intracellular Mg 2+ level, by competitively gating Ca 2+ channels, can attenuate Ca 2+ influx [68], which can rapidly reduce ΔΨ [69]. Once the ΔΨ of mitochondria is increased the mitochondria are more likely to undergo anterograde transport [70], which might partially account for our findings that the mitochondrial density in distal branches increased (Figs. 6-8). However, the exact mechanisms need further elucidation.
Finally, our previous work demonstrates the important role of Mg 2+ as a positive regulator of NR2B-containing NMDARs in controlling synaptic plasticity [24]. In the current study, we revealed the molecular mechanisms of Mg 2+ in regulating presynaptic functional terminal density (Fig. 9), and found that sufficiently high [Mg 2+ ] i might be essential for maintaining a high density of functional presynaptic terminals, thereby sustaining a high sensitivity of synaptic networks for information transmission. These studies provide the mechanistic understanding of how elevating brain Mg 2+ concentration can prevent age-related memory decline in old rats [62], reverse cognitive decline in Alzheimer's Disease (AD) model mice [71] as well as ameliorate cognitive decline in subjects with mild cognitive impairment (MCI) (GL et al. manuscript in submission).

Imaging and analysis
Most of the in vitro fluorescent or differential interference contrast (DIC) images for an area of interest (AOI) (59 μm × 59 μm) were taken at 1024 × 1024 pixels with a 0.05754 μm/pixel resolution using a 60X NA1.20 water-immersion objective on a Laser Scanning Confocal Microscopy System FV300 (Olympus) at room temperature (RT, 22-26°C), the exceptions were clarified in the following methods. A z-stack of images was taken with 0.5-1 μm steps and compressed at maximal intensity to generate the final image, which was then processed and analyzed in Image-Pro Plus 5.0 (IPP5.0) (Media Cybernetics) and/or an open source software Fiji (Image J). For the measurements of vesicle turnover and protein expression in terminals at dendrites, the AOIs were chosen at the high-density regions of dendritic branches among the "islands" of cell bodies. The general criterion for choosing a "high-density dendritic area" was that most of area in the AOI was covered by neuronal branches. For the fluorescent colocalization experiments (described below), the images from the same AOI were aligned together and registered using the algorithms Rigid Body Registration [72] and UnwarpJ [73] to register the images and tackle the distortions in Fiji. For in vivo experiments, the Electron Microscopy (EM) images and immunofluorescent images of ultrathin brain slices were described below.

Hippocampal neuron cultures
High-density primary cultures of hippocampal CA3-CA1 pyramidal neurons of neonatal Sprague-Dawley rats (<24 hr) were used in the current study, as described Fig. 9 Regulation of functionality of a terminal by intracellular Mg 2+ . Schematic cartoon to illustrate the regulation of presynaptic functionality at a single terminal: ↑[Mg 2+ ] i ⇒ ↑Mitochondria ⇒ ↑[ATP] i ⇒ ↑Axonal transport efficiency ⇒ ↑Ca 2+ -sensitivity-related proteins ⇒ ↑Ca 2+ sensitivity ⇒ Terminal in functional state before [24]. The neurons were cultured on 1# coverslips (8 mm × 8 mm) for 14-28 div before use. [Mg 2+ ] o in culture medium was adjusted to different concentrations according to experimental designs. In each experiment, sister cultures from the same batch were always used for treatment and control conditions on the same day, and the experimental results were repeated using several different batches.
Vesicle turnover detection FM dyes were used to visualize the vesicle turnover of presynaptic terminals as previously described [24]. In the current study, 10 μM FM1-43 or 20 μM FM4-64 (Biotium) was loaded under different patterns of field stimulations, such as 5AP bursts (5 APs at 100Hz for each burst, 6 bursts (30 APs totally) with 10 s inter burst interval were given) and "maximal stimulation" (600 APs at 10 Hz) [54]. Releasable fluorescence in the AOI (ΔF) was obtained from the difference of loading (F 1 ) and unloading (F 2 ) images, i.e. ΔF = F 1 -F 2 (see Fig. 1a). For Fig. 3c, the loading/unloading procedures were performed under 5AP bursts and 600AP stimulation at the same area of network successively. Subsequently, the ΔF 5AP and ΔF 600AP images were registered to colocalize the vesicle turnover for these two stimulating protocols for individual terminals.

Functional terminal density measurement
The functional terminal density (N 5AP ) at a dendritic branch was defined as the number of 5AP bursting stimulation induced FM(+) puncta (#FM 5AP ) per unit area of dendritic branch (A), N 5AP = #FM 5AP /A. Retrograde immunofluorescence (IF) staining of MAP2 (as described below), which specifically presents in dendritic skeleton, was used to label dendritic area and A was calculated from MAP2(+) area. To limit the error of A introduced by the immunoreactivity of MAP2(+), the staining and imaging parameters were rigorously controlled in different batches of IF and the grey-scale histograms of MAP2(+) images were equalized before measuring A. Here, to ensure the equalized MAP2(+) marked area could reflect the real morphology of dendrites, the parameters of histogram equalization were set based on the comparison of MAP2(+) and DIC images. For most experiments, the average N 5AP of an AOI was calculated from the total number of FM(+) puncta divided by total MAP2(+) area to reduce the sampling error. Then the mean N 5AP of all AOIs (1-5 AOIs/coverslip) was used to represent the average N 5AP in a coverslip; for each statistical data point, several coverslips were used and the mean ± SEM of all coverslips was represented (see Figure  legends). For Fig. 3b and d, the FM(+) puncta density induced by 600AP stimulation was also calculated and presented in the same way as described above. Exceptionally, for Fig. 2b, each point represented the N 5AP at a single segment of dendritic branch. In general, in an AOI (59 μm × 59 μm, with varying dendritic density) 351-1598 presynaptic functional terminals could be detected by FM dyes for different experimental groups under 5AP bursting stimulation.
The following primary and secondary antibodies were

In vitro protein immunofluorescence quantifications
For Fig. 3b, the average density of immunoreactive puncta of a given AOI was calculated from the total puncta number divided by the total MAP2(+) area; the values from several AOIs were averaged to represent the average puncta density in a coverslip; for each data bar, the mean ± SEM of these coverslips were presented. For Figs. 4d, 5d-f, the total fluorescence intensity of all immunoreactive puncta in an AOI was divided by the total MAP2(+) area to get the average immunoreactivity per unit area of dendrites in the AOI; several AOIs were measured from a coverslip and the mean of these AOIs was calculated to represent the average immunoreactivity per unit area of dendrite in the coverslip; for each data point, several coverslips were used and the mean ± SEM of these coverslips were presented. For Fig. 5c and d, the mean fluorescence intensity per pixel in each cell body was measured (the somatic area of each cell body was measured from DIC image) and then the mean ± SEM of all the cell bodies were presented. For Fig. 7b-e, the average protein immunoreactivity of an AOI was defined as the total fluorescence of a protein per unit area of MAP2(+) marked dendrites (as described above); 9 AOIs were selected from a low [Mg 2+ ] o (Ctrl 0.8) coverslip or a high [Mg 2+ ] o (1.2 4hr) coverslip respectively (these two coverslips were sister cultures from the same culturing dish); for each protein, the average protein immunoreactivity values were normalized to the mean of the 9 AOIs of Ctrl 0.8 group. We defined the normalized immunoreactivity per unit area of dendrites as Q index, then ΣQ proteins = Q SYT1 + Q Rab3a + Q RIM1 + Q Munc13 + Q ELKS + Q Syntaxin1 .

Intracellular Mg 2+ measurement
The mature cultures were incubated in Tyrode solution with 2 μM Magnesium Green-AM (MgGrn, an intracellular Mg 2+ indicator, Invitrogen) for 30 min and washed out for 20 min; both at 37°C. Fluorescent images of several AOIs in each coverslip were taken on FV300 at RT. In the current study, we measured the MgGrn fluorescence at basal condition (without eliciting any AP stimulus) (Fig. 1a) to eliminate any effects caused by AP-induced intracellular Ca 2+ fluctuations. Since the intracellular Ca 2+ concentration ([Ca 2+ ] i ) at basal condition was usually < 100 nM, the contribution of Ca 2+ to MgGrn fluorescence was very small [74]. Thus, the fluorescent intensity of MgGrn could be considered proportional to [Mg 2+ ] i . Since the measured MgGrn fluorescence was also dependent on the local volume of branches in network, we built a physical model to describe this phenomenon and derived the calculating formula that corrected the effect of varying local volumes.
At basal condition, the total fluorescence in a branch was positively proportional to the total amount of free Mg 2+ ions in cytoplasm. We assumed a cylindrical shape of each neuronal branch (Additional file 1: Figure S1. A); thus we could deduce the following relationship: Where fluo total was the total fluorescence, F(x, y, z) was the spatial distribution function of fluorescence, dv was the differential volume at the point (x, y, z), V' represented the volume of fluorescence detectable space.
Then we derived the relationship as follows based on the assumption that [Mg 2+ ] i was proportional to integral fluorescence per unit volume of a neuronal branch: Fðx; y; zÞdxdydz V Where V was the volume of the branch. From Additional file 1: Figure S1. A and B we could further derive that: Where [−h, h] was the range in z-direction, where fluorescence was detectable, L, d were the length (in xdirection) and width (in y-direction) of the maximal projection of fluorescence on xy-plane (Additional file 1: Figure S1. B), which were roughly equal to the length and diameter of the branch when the threshold of fluorescence during segmentation was set properly. Thus the branch volume V was approximately equal to πd 2 L/4.
We set θ (μm/pixel) as the resolution in x-and y-direction, which was 0.05754 μm/pixel in our experiments, and set ρ as the resolution in z-direction, which was equal to the scanning step of z-stack, i.e. ρ = 1 μm. Thus L = lθ, d = mθ, 2h = nρ, where l, m were the pixels of the branch in x, y directions, n was the number of images in the stack. Then we got: Where FðzÞ was the mean fluorescence per pixel in the region of the branch in the image (whose coordinate was z) in z-stack (Additional file 1: Figure S1. B).
Since we observed that in the range of [−nρ/2, nρ/2], FðzÞ exhibited well Gaussian distribution (Additional file 1: Figure S1. C), and the normalized distribution curve was the same among different branches (Additional file 1: Figure S1. C), then we knew ΣFðzÞ was linearly proportional to FðzÞ max according to the properties of Gaussian curve, thus: And we could further simplify this formula as follows: Where FðzÞ max was the mean intensity per pixel in the area of a branch in the compressed image of the stack (the compressed image was achieve by maximal z-projection of the stack, as described above). From the formula, we knew that [Mg 2+ ] i was approximately positively proportional to FðzÞ max but negatively proportional to the diameter of branch, consistent with the experimental observations (Additional file 1: Figure S1. D and E).
In the experiments, given a randomly selected AOI of dendritic area (the image was obtained as described above), 50-100 branches were randomly selected from each AOI and the mean intensity of fluorescence (i.e.

Mitochondrial status assessment
For Fig. 7a, mitochondria were marked with 40 nM Mitoview633 (Biotium) for 15 min and then washed out in blank medium for 15 min, both at 37°C. For Fig. 6a, the membrane potential-sensitive fluorescent dye JC-1 (1 μM) (Invitrogen) was used. JC-1 was incubated for 15 min followed by a 15 min washout at 37°C. Fluorescent images were taken on FV300 confocal. Mitochondrial membrane potential (ΔΨ) of each mitochondrion was estimated by the ratio of fluorescence of aggregate versus monomer form of JC-1, i.e. F aggr /F mono [61]. JC-1 was excited by 488 nm laser and the emission spectra was collected at 510-575 nm and > 575 nm at the same time separately. Then we calculated the ratio of fluorescence at > 575 nm versus at 510-575 nm (F aggr /F mono ) for each individual mitochondrial punctum to represent the ΔΨ of that mitochondrion. To estimate the valid range of F aggr /F mono , we measured F aggr /F mono of individual mitochondria before and after administering 5 μM FCCP 5 min at the same AOI. The mean F aggr /F mono of all the mitochondria (1.21 ± 0.02, n = 942 mitochondria) was lowered to 0.26 ± 0.002 (the minimal value of F aggr / F mono ). Thus, we considered the F aggr /F mono value of a mitochondrion to be meaningful only if it was > 0.26 in our experiments, and the puncta with F aggr /F mono < 0.26 (17/942 mitochondria in this experiment, i.e.~2 %) were excluded in statistics. F aggr /F mono was calculated for each individual meaningful punctum and the mean value of population was calculated from all the puncta to represent the general ΔΨ in this AOI. 3-5 AOIs were measured for each coverslip and the mean of the AOIs were calculated to represent the average ΔΨ of the coverslip. For each bar or point in Fig. 6, several coverslips were used (described in legends). We also quantified the density of mitochondria (N mito ) by counting the number of JC-1-aggregate(+) fluorescent puncta (Fig. 6) or Mito-View633(+) puncta (Fig. 7) per area of branches. We observed no difference in N mito measurement between the two markers.

Co-staining of multiple fluorescent markers
For Fig. 3a, FM1-43 staining was performed and images were taken on confocal. Then retrograde IF was performed at the same AOI. For Fig. 7a, MitoView633 was used to mark mitochondria, images were taken, and then FM1-43 staining was performed and images were taken. For the combination of FM and IF imaging, a new strategy of retrograde IF was performed for several rounds to label multiple presynaptic proteins at a given AOI (HZ and GL unpublished method: Single Synapse Analysis by FM1-43 and Immunofluorescence Imaging Array, which we named SAFIA). For each round, 2-3 antibodies were stained and probed by fluorophore-conjugated secondary antibodies (as described above), and then both the DIC and the fluorescent images were taken immediately. After taking all images, primary and secondary antibodies were completely eluted (data not shown) by a stripping buffer containing 0.2 M NaOH and 0.015 % (w/ v) SDS (Amresco) in deionized water for 20 min RT, twice, and then washed out gently and thoroughly in Tris buffer (described above) for more than 1 hr RT (to make sure no residual SDS was left). Then the IF of other 2-3 primary antibodies at the same AOI was performed as described above and images of these AOIs were taken. DIC image was used as landmark of each AOI. All the fluorescent images were aligned and registered in Fiji and analyzed as described above. In the first round of IF in the SAFIA experiments, 300 μM ADVASEP-7 (Biotium) was added into the blocking solution during the permeablization procedure to quench the residual FM-dye in the membrane and to ensure that the background introduced by FM-dye was as low as possible in the following IF experiments (data not shown). For Figs. 1b and 2a, [Mg 2+ ] i was stained with MgGrn, images were taken at basal condition, and then the FM4-64 staining was performed. All the procedures were described separately as aforementioned.

Western blot
[Mg 2+ ] o was elevated from 0.8 to 1.2 mM in mature sister cultures (14-28 div) for 6 hr. Proteins were extracted from the cultures and then resolved on Polyacrylamide gels. Proteins were transferred from gels to PVDF membrane and probed with the following antibodies: anti-SYP (Millipore) 1:50,000, SYT1 1:5,000, Rab3a 1:500-1,000, RIM1 1:800, Munc13-1 1:400, ERC1b2 (ELKS) 1:1,000, Syntaxin1 1:500-1,000 (SYSY), GAPDH 1:1,000 (CST). The other procedures were the same as previously described [62]. For the analysis, digital images were quantified using IPP5.0, and the level of each protein was normalized by the level of GAPDH in the same lane. The sample from each coverslip was resolved individually and repeated for 3 times, and then the average value of the 3 times was calculated to represent the protein level in the coverslip. For each bar in Fig. 5a, the mean ± SEM of coverslips was presented.

ATP measurement
Cultured neurons were quickly detached from a coverslip with 500 μL boiling extraction solution [75] containing 40 mM HEPES (pH 7.8) and 4 mM MgSO 4 . The solution was repeatedly pipetted to ensure complete lysis. The lysed neurons were immediately transferred into a centrifugal tube and centrifuged at 12,000 rpm at 4°C for 5 min. For each coverslip, total ATP content of supernatant was immediately determined using a Luciferin-luciferase ATP Assay Kit (Invitrogen). For each data point in Figs. 6c, 7e and f, the mean ± SEM of coverslips was presented.
For the blocks prepared for EM, they were post-fixed in 1 % Osmium Tetraoxide (Ted Pella) and 1.5 % Potassium Ferricyanide (Sigma) in MB (pH 6.0) for 1 hr, and stained with 3 % Uranyl Acetate (Ted Pella) for 1 hr. Then the blocks were dehydrated with 50, 70, 80, 95 %, 100 %, and 100 % Ethanol sequentially, for 15 min each. Following dehydration, blocks were placed in 100 % Propyleneoxide (Sigma) for 10 min twice. All the procedures above were performed on ice. Then blocks were infiltrated with 50 % Propyleneoxide + 50 % SPI-PON 812 resin (SPI-CHEM) for 60 min RT, 100 % resin for 24 hr at 4°C, and subsequently transferred into embedment molds fulfilled with pure resin and placed in 60°C oven for polymerization for 24 hr. 70 nm ultrathin slices were cut and then stained with 3 % Uranyl Acetate and 0.4 % (w/v) Lead Citrate, sequentially. Images were taken on Transmission (Hitachi H-7650) (80 kV, 1k × 1k pixels, 25kX, 2 nm/pixel) or Scanning EM (Supra55, Zeiss) (10 kV, 8k × 8k pixels, 2 nm/pixel, 15 μs/pixel). For the SEM, ultrathin slices were mounted on silicon chips, which can enhance the electro-conductivity dramatically (even without carbon coating). The density of mitochondria in an EM image was defined as #mitochondria/area. For each rat, the density of mitochondria was calculated from the mean density of 67-96 TEM images and 5 SEM images (the density measured in TEM images was the same as that measured in SEM images).
For the blocks prepared for IF, they were treated with 1 % Tannic Acid (Sigma) for 1hr, 1 % Uranyl Acetate in Maleate Acid buffer (pH 6.0) for 1hr, and then dehydrated with 50, 70, 80, 95, 100 %, and 100 % Ethanol containing 1 % PPD (Sigma) sequentially, for 15 min each. Mixture of Ethanol and LR White resin (E.M.S.) with the ratios 2:1, 1:1, 2:1 and pure resin were used to infiltrate the tissue blocks, each for 1 hr, then replaced by 100 % LR White for 24 hr. All the above procedures were performed on ice. The blocks were placed in capsules filled with LR White resin and put in 55°C oven for polymerization for 48 hr. 70 nm ultrathin slices were cut and mounted on coverslips. The IF was performed according to Array Tomography technique [76]. SYP, VGLUT1, SYT1, Rab3a, RIM1, Munc13-1, ELKS and Syntaxin1 were stained in the ultrathin slices and fluorescent images of each protein were taken at 1024 × 1024 resolution with 0.03836 μm/pixel resolution using a 60X objective (UPlanSApo 60XW N.A. 1.20) on FV300. For each protein, the total fluorescence (F) of all the puncta per area of image was measured to represent the quantity of that protein. For each rat, F values from 5-7 tissue blocks were averaged to represent the protein quantity in that rat (F rat ). We normalized F rat of each protein by the mean value of all rats in control group to get the normalized quantity Q. To estimate the total quantity of the Ca 2+ -sensitivity-related proteins, we summated the Q of the 6 proteins in the same rat together to represent the total quantity in that rat (ΣQ proteins ), ΣQ proteins = Q SYT1 + Q Rab3a + Q RIM1 + Q Munc13 + Q ELKS + Q Syntaxin1 .

Experimental animals
All the rats involved in this paper were purchased from Vital River Laboratory (Beijing). All the animal experiments were approved by Tsinghua University Committees on Animal Care.

Statistics analysis
Data are shown as mean ± SEM. Statistical significance is considered as p < 0.05. Two-tailed Student-t or Kolmogorov-Smirnov test is used.

Additional file
Additional file 1: Figure S1.