1. Neuroscience
Download icon

Spontaneous neurotransmission signals through store-driven Ca2+ transients to maintain synaptic homeostasis

  1. Austin L Reese
  2. Ege T Kavalali  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
Research Article
  • Cited 20
  • Views 1,878
  • Annotations
Cite as: eLife 2015;4:e09262 doi: 10.7554/eLife.09262

Abstract

Spontaneous glutamate release-driven NMDA receptor activity exerts a strong influence on synaptic homeostasis. However, the properties of Ca2+ signals that mediate this effect remain unclear. Here, using hippocampal neurons labeled with the fluorescent Ca2+ probes Fluo-4 or GCAMP5, we visualized action potential-independent Ca2+ transients in dendritic regions adjacent to fluorescently labeled presynaptic boutons in physiological levels of extracellular Mg2+. These Ca2+ transients required NMDA receptor activity, and their propensity correlated with acute or genetically induced changes in spontaneous neurotransmitter release. In contrast, they were insensitive to blockers of AMPA receptors, L-type voltage-gated Ca2+ channels, or group I mGluRs. However, inhibition of Ca2+-induced Ca2+ release suppressed these transients and elicited synaptic scaling, a process which required protein translation and eukaryotic elongation factor-2 kinase activity. These results support a critical role for Ca2+-induced Ca2+ release in amplifying NMDA receptor-driven Ca2+ signals at rest for the maintenance of synaptic homeostasis.

https://doi.org/10.7554/eLife.09262.001

eLife digest

Learning and memory is thought to rely on changes in the strength of the connections between nerve cells. When an electrical impulse travelling through a nerve cell reaches one of these connections (called a synapse), it causes the cell to release chemical transmitter molecules. These bind to receptors on the cell on the other side of the synapse. This starts a series of events that ultimately leads to new receptors being inserted into the membrane of this second cell, which strengthens the connection between the two cells.

The receptors involved in this process belong to two groups, called AMPA and NMDA receptors. Both groups are ion channels that regulate the flow of charged particles from one side of a cell's membrane to the other. In resting nerve cells, NMDA receptors are partially blocked by magnesium ions. However, the binding of the transmitter molecules to AMPA receptors causes these receptors to open and allow positively charged sodium ions into the cell. This changes the electrical charge across the cell membrane, which displaces the magnesium ions from the NMDA receptors so that they too open. Calcium ions then enter the cell through the NMDA receptors and activate a signaling cascade that leads to the production of new AMPA receptors.

Nerve cells also release transmitter molecules in the absence of electrical impulses, and evidence suggests that individual cells can use this ‘spontaneous transmitter release’ to adjust the strength of their synapses. When these spontaneous release levels are high, AMPA receptors are removed from the membrane of the nerve after the synapse to make it less sensitive to the transmitter molecules. Conversely, when spontaneous release levels are low, additional AMPA receptors are added to the membrane to increase the sensitivity.

Reese and Kavalali have now identified the mechanism behind this process by showing that spontaneously released transmitter molecules cause small amounts of calcium to enter the second nerve cell through NMDA receptors, even when these receptors are blocked by magnesium ions. This trickle of calcium triggers the release of more calcium from stores inside the cell, which amplifies the signal. The ultimate effect of the flow of calcium into the cell is to block the production of AMPA receptors, and ensure that the synapse does not become any stronger. As confirmation of this mechanism, Reese and Kavalali showed that simulating low levels of spontaneous activity by blocking the so-called ‘calcium-induced calcium release’ has the opposite effect. This led to more AMPA receptors being produced and stronger synapses. Taken together these findings indicate that spontaneous transmitter release exerts an outsized influence on communication between neurons by maintaining adequate levels of AMPA receptors via these ‘amplified’ calcium signals.

https://doi.org/10.7554/eLife.09262.002

Introduction

Studies in the last decade have shown that spontaneous release events trigger biochemical signaling leading to maturation and stability of synaptic networks, local dendritic protein synthesis and control postsynaptic responsiveness during homeostatic synaptic plasticity (Chung and Kavalali, 2006; Sutton et al., 2006; Kavalali, 2015). Most surprisingly, these studies have demonstrated that postsynaptic excitatory receptor blockade or inhibition of neurotransmitter release in addition to action potential blockade induces faster and more pronounced homeostatic synaptic potentiation (Sutton et al., 2006; Nosyreva et al., 2013). There is evidence that alterations in resting Ca2+ signaling, partly triggered via activation of NMDA receptors at rest, is critical for these effects (Wang et al., 2011; Nosyreva et al., 2013). However, to date there is no direct information on the properties of dendritic Ca2+ signals elicited by spontaneous release events under physiological circumstances. Our group's previous work as well as others used electrophysiology to show that, indeed, under physiological levels of extracellular Mg2+ spontaneous miniature excitatory postsynaptic currents (mEPSCs) possess a sizable NMDA receptor-mediated component, indicating that NMDA receptors signal at rest under physiological conditions without requiring local AMPAR-mediated dendritic depolarizations (Espinosa and Kavalali, 2009; Povysheva and Johnson, 2012; Gideons et al., 2014). The existence of an NMDA component within mEPSCs agrees with earlier estimates of incomplete Mg2+ block of canonical NMDA receptors near resting membrane potentials, and therefore it does not necessarily involve NMDA receptor subunits with altered Mg2+ sensitivity (Jahr and Stevens, 1990). Nevertheless, the NMDA receptor Ca2+ influx under these conditions is estimated to be small, corresponding to approximately 20% of the full Ca2+ influx carried by unblocked NMDA receptors (Espinosa and Kavalali, 2009). Therefore, as NMDA receptor-driven Ca2+ signals at rest are expected to be relatively minor in magnitude, it remains unclear how their blockade could be critical in producing homeostatic synaptic scaling. To address this question, we visualized the resting NMDA receptor-driven Ca2+ signals and found that they are amplified by a Ca2+-induced Ca2+ release mechanism to elicit downstream signaling events. Importantly, based on this information, we also show that direct suppression of these resting Ca2+ signals is sufficient to elicit eEF2 kinase dependent postsynaptic scaling.

Results

Visualization of miniature spontaneous Ca2+ transients in hippocampal neurons

To detect transient Ca2+ signals that occur under resting conditions—in the absence of action potentials—we took advantage of the Ca2+ indicator dye Fluo-4 or the Ca2+ sensitive fluorescent protein GCaMP5K as reporters (Gee et al., 2000; Akerboom et al., 2012). To visualize synapses, both reporters were used on hippocampal neurons that were infected with lentivirus expressing the fusion protein Synaptobrevin2-mOrange (Syb2-mOrange) consisting of a chimera of the synaptic vesicle protein synaptobrevin2 with the pH sensitive red-shifted fluorophore mOrange (Ramirez et al., 2012) (Figure 1A–D). In these experiments, the signal contribution of Syb2-mOrange during live imaging is negligible (see Figure 2—figure supplement 1). In Fluo-4 experiments, neurons were initially incubated and labeled with the membrane permeable analog of Fluo-4 (Fluo-4 AM) (Figure 1A) followed by dye removal and perfusion with a Tyrode's solution containing 2 mM Ca2+, 1.25 mM Mg2+ as well as 1 μM tetrodotoxin (TTX) to block action potentials. Fluorescence images were collected at a frequency of 10 Hz and fluorescence intensity traces were generated for the regions of interest (ROIs) selected over Syb2-mOrange puncta which fluorescence was maximized at the end of each experiment using 50 mM NH4Cl (Figure 1E). Under these conditions, we could detect rapid Ca2+ transients (miniature spontaneous calcium transients or mSCTs) with absolute values that were at least 2 standard deviations above the mean of the preceding baseline period (2 s) (Figure 1F). These events occurred at a frequency of 0.32 ± 0.04 min−1 per ROI, consistent with earlier estimates of the frequency of spontaneous fusion events per release site (Leitz and Kavalali, 2014). Repeating the same experimental protocol with Fluo-4 AM in the absence of Mg2+ did not yield a significantly different mSCT frequency (Figure 1F) suggesting that under physiological Mg2+ concentrations we could detect a majority of mSCTs. Interestingly, even though the presence of extracellular Mg2+ is expected to greatly diminish NMDAR current magnitudes (Espinosa and Kavalali, 2009; Gideons et al., 2014), imaging experiments did not reveal a significant difference in mSCT amplitudes detected in Mg2+ (1.25 mM Mg2+ ∆F/Fo = 0.063 ± 0.001, 0 mM Mg2+ ∆F/Fo = 0.067 ± 0.002, p = 0.16, Student's unpaired t-test, N = 825 events from 8 experiments). The fact that mSCT amplitude was unaffected by extracellular Mg2+ indicates mSCTs measured by Fluo-4 AM were not likely to be solely dependent on NMDA receptor activity.

Multiple approaches to detect miniature spontaneous Ca2+ transients (mSCTs) in the presence of TTX and physiological Mg2+.

(A) Loading dissociated rat hippocampal cultures with Fluo-4 AM dye labels all cells on the coverslip (above) and produces the largest signal amplitudes, shown as example traces and an average with standard deviation (below) N = 38 experiments, 7 cultures. (B) Individual neurons were loaded with the salt form of Fluo-4 at the whole cell recording configuration via a pipette containing 200 μM of the dye. N = 4 experiments, 1 culture. (C) Low efficiency lipotransfection with the highly sensitive GCaMP5K variant produces sparse labeling of neurons across the coverslip but low signal (ΔF/F) amplitudes. N = 5 experiments, 1 culture. (D) Lentiviral mediated transfection with GCaMP5K-PSD95 targets the fluorescent construct to the postsynaptic densities of all cells on each coverslip. N = 15 experiments, 4 cultures. (E) Example images and trace of a mSCT visualized with Fuo-4 AM and its corresponding Syb2-mOrange puncta. Panels show baseline and peak fluorescence intensity with the arrow marking peak fluorescence intensity of the mSCT. Scale bar 5 µm. (F) Frequencies expressed as mSCTs per ROI per minute show the highest efficiency of mSCT detection with Fluo-4 AM. Fluo-4 AM based experiments performed with no Mg2+ in the external solution reported no significant changes in mSCT compared to the presence Mg2+. The postsynaptically localized reporter GCaMP5K-PSD95 reports statistically lower frequencies when compared to Fluo-4 AM (Fluo-4 AM 1.25 mM Mg2+ vs GCaMP5K-PSD95 1.25 mM Mg2+ p = 0.0003, Fluo-4 AM 0 mM Mg2+ vs GCaMP5K-PSD95 1.25 mM Mg2+ p = 0.0031, via one-way ANOVA with Holm-Sidak's multiple comparisons) 0 mM Mg2+ Fluo-4 AM N = 16 experiments, 8 cultures. 0 mM Mg2+ GCaMP5kK-PSD95 N = 10 experiments, 4 cultures.

https://doi.org/10.7554/eLife.09262.003

In parallel experiments, we delivered the salt form of Fluo-4 (200 µM) with a patch pipette in the whole-cell recording configuration and performed the same imaging protocol as above (Figure 1B). In this setting, we detected a lower frequency of events (0.125 ± 0.035 min−1 per ROI), indicating that some of the mSCTs may be susceptible to postsynaptic dialysis and wash out of soluble factors (Figure 1F). In agreement with this premise, when the same optical recording conditions were applied to neurons expressing a soluble version of the green emission Ca2+ indicator probe GCaMP5K, we could detect a higher frequency of mSCTs (0.230 ± 0.04 min−1 per ROI).

In subsequent experiments, we expressed a fusion construct of GCaMP5K with the postsynaptic scaffolding protein PSD95 (GCAMP5K-PSD95) in order to target the calcium sensor specifically to the postsynaptic density (Figure 1D). In the presence of extracellular Mg2+ based on the population average this setting provided the lowest estimate for the mSCT frequency (0.009 ± 0.004 min−1 per ROI) (Figure 1F). In contrast, removal of Mg2+ augmented the mSCT detection rate to a level comparable to the rates we observed with Fluo-4 or soluble GCaMP5K (Figure 1E). This finding suggests that, in the presence of Mg2+, postsynaptically localized GCaMP5K-PSD95 has limited ability to detect the Ca2+ signals generated in its vicinity via Ca2+ influx. However, experiments in the absence of Mg2+ indicate that this probe is functional and can in principle detect these spontaneous local Ca2+ transients as reported earlier (Leitz and Kavalali, 2014).

Recording in the presence of 1.25 mM Mg2+ and 1 µM TTX, we could detect spontaneously generated Ca2+ transients in the dendrites of hippocampal pyramidal cells with all four techniques. Although, each probe reports a different frequency these differences are statistically insignificant except when considering the difference between Fluo-4 AM and GCaMP5K-PSD95 (Figure 1F). Relatively lower detection efficiency of GCAMP5K-PSD95 compared to soluble probes illustrates that the majority of these transients are not localized to the postsynaptic density. The failure of Mg2+ to decrease mSCT amplitudes as measured with Fluo-4 AM strongly suggest that a majority of transients are generated by a signaling process downstream of Ca2+ entry rather than reporting the Ca2+ influx per se. In order to identify the nature of this signaling, in subsequent experiments, we used the Fluo-4 AM based imaging to test conditions that alter mSCTs.

The generation of mSCTs requires NMDA receptor mediated Ca2+ influx

To characterize mSCTs, neurons were labeled with Fluo-4 AM as in Figure 1A and imaged in Tyrode's solution containing TTX (Figure 2A). Ca2+ transients were detected by the slope of the rising phase as well as the peak amplitude. To ensure that these detected peaks were not noise, only mSCTs with a peak amplitude 2 standard deviations greater than the signal average of the previous 2 s were counted. Figure 2B shows the rise and decay times as well as the fluorescence amplitudes of 306 mSCTs identified from 6 experiments. In these experiments, the mean rise time was 0.38 s with a median of 0.29 s. The mean decay time was 0.86 s with a median of 0.47 s. The amplitude distribution had an average ∆F/Fo of 0.061 with a median of 0.049 (Figure 2B).

Figure 2 with 1 supplement see all
Detection and characterization of spontaneous Ca2+ transients in physiological concentrations of Mg2+.

(A) Events detected from Fluo-4 AM traces having rising slope greater than 350 fluorescence units/s and a peak ∆F/Fo greater than 0.035 were counted if the peak fluorescence value was 2 standard deviations greater than the mean of the signal 2 s previous. Gray shaded region indicates the moving average plus/minus two standard deviations and the red line indicates the 0.035 ∆F/Fo threshold. Red trace shows the 2 point slope with the black line as the 350 A.U./second detection threshold. Arrows indicate peaks that satisfy these criteria. (B) Histograms showing rise time (τ), decay time (τ) and amplitudes (∆F/Fo) of mSCTs. N = 306 mSCTs from 6 experiments and 2 cultures. (C, D) Traces from cells (C) and Ca2+ transient frequencies (D) were obtained by imaging first in Tyrode's solution containing no Ca2+, then in Tyrode's containing 2 mM Ca2+ and finally Tyrode's containing 2 mM Ca2+ and the NMDA receptor blocker AP5. Removal of extracellular Ca2+ or block of the NMDA receptor resulted in a significant reduction in Ca2+ transient frequency (2 mM Ca2+ vs 0 mM Ca2+ p = 0.038, 2 mM Ca2+ vs 2 mM Ca2+ + AP5 p = 0.038, via 1-way ANOVA with Holm-Sidak's multiple comparisons test). N = 8 experiments, 2 cultures.

https://doi.org/10.7554/eLife.09262.004

Next we tested whether NMDA receptor activity is required for the generation of mSCTs. For this purpose, synaptic ROIs were imaged in three steps. First optical recordings were obtained in Tyrode's solution with nominal Ca2+ containing 1 µM TTX followed by the addition of 2 mM Ca2+ and finally in Tyrode's solution containing TTX + 2 mM Ca2+ + 50 µM AP5 (Figure 2C). In the absence of Ca2+ in the bath, mSCTs were virtually undetectable suggesting that Ca2+ influx is required for their generation. Switching the Tyrode's solution to TTX + 2 mM Ca2+ brought the mSCT frequency back to normal levels, and subsequent addition of the NMDA receptor antagonist AP5 again decreased the mSCT frequency to very low levels that were not statistically different from the nominal Ca2+ condition (Figure 2D). These results indicate that Ca2+ influx through the NMDA receptor is critical for the generation of mSCTs.

mSCTs are driven by spontaneous neurotransmitter release

To examine whether the NMDA receptor openings driving mSCTs were due to spontaneous glutamate release we took two complementary approaches. First, we took advantage of the fact that the acute application of 100 mM hypertonic sucrose is known to produce an increase in mEPSCs (Fatt and Katz, 1952; Rosenmund and Stevens, 1996). To measure this effect, hippocampal pyramidal cells were voltage clamped at −70 mV while a baseline AMPA mEPSC frequency was collected in Tyrode's solution containing 1 µM TTX, 50 µM PTX and 50 µM AP5 for 2 min. Perfusion was then switched to Tyrode's containing 100 mM hypertonic sucrose as the recording continued for 2 min. Quantification of these recordings revealed a 2.6-fold increase in mEPSC frequency upon the addition of hypertonic sucrose (Figure 3A). To test whether the increase in mEPSC frequency could drive an increase in mSCT frequency the experiment was repeated in neurons loaded with Fluo-4 AM. The baseline was collected in Tyrode's solution containing only TTX before changing to a solution containing TTX + 100 mM sucrose. The addition of hypertonic sucrose produced a 2.3 fold increase in mSCT frequency (Figure 3B), which supports the hypothesis that spontaneous glutamate release can drive the generation of postsynaptic calcium transients.

mSCT frequency is correlated with mEPSC frequency.

(A) Whole cell recordings from WT cells (left) show a ∼twofold increase in mEPSC frequency when switched to Tyrode's solution containing 100 mM sucrose (right) (p = 0.002, Student's paired T test, N = 8 cells, from 5 coverslips and 2 cultures). (B) Example traces (left) and quantification of spontaneous Ca2+ transient frequencies measured via imaging show a ∼twofold increase upon application of 100 mM sucrose (right) (p = 0.028, Student's paired T test, N = 9 experiments from 3 cultures). (C) Fluo-4 example traces from both control and SNAP25 KO animals before and after the application of AP5. (D) Fluo-4 imaging in cultures made from SNAP25 KO and littermate control mice reveal that the KO cultures have a substantially decreased mSCT frequency. In this setting, AP5 treatment greatly decreases but does not completely abolish the remaining mSCTs. (WT, TTX vs WT, TTX+AP5 p = 0.010. WT, TTX vs KO, TTX p = 0.008. KO TTX vs KO TTX+AP5 p = 0.003, via 1-way ANOVA with Tukey's multiple comparisons, N = 8 experiments in WT cells and 9 experiments in KO cells from 3 cultures).

https://doi.org/10.7554/eLife.09262.006

Next, to assess whether a decrease in mEPSC frequency would correlate with a decrease in mSCT frequency, we utilized neurons from mice lacking the critical SNARE-mediated fusion machinery component SNAP-25 (Washbourne et al., 2002). These mice die at birth; however, hippocampal neurons cultured from embryonic mice form synapses and manifest a virtual absence of evoked neurotransmission and highly diminished rate of spontaneous neurotransmitter release (Bronk et al., 2007). In Fluo-4 AM imaging experiments with hippocampal cultures made from littermate control mice, the application of AP5 was able to produce a significant decrease in mSCT frequency compared to baseline recorded in TTX, as had been observed previously in wild-type rat cultures. Neurons derived from SNAP25 knock out animals had a significantly decreased baseline mSCT frequency. Also, these transients remained sensitive to AP5, which is again consistent with mSCT generation being driven by spontaneous vesicle release (Figure 3C,D).

mSCTs do not require activation of AMPA receptors, L-type Ca2+ channels, or group I mGluRs

Mature glutamatergic synapses contain both AMPA and NMDA receptors (Bekkers and Stevens, 1989; Liao et al., 2001). Therefore, in the next set of experiments we tested whether concurrent AMPA receptor activity augments NMDA receptor activity at rest through electrical means. Such synergy between the activation of the two types of receptors may be facilitated by dendritic spines that possess a high spine neck resistance that render them electrically isolated from the dendritic shaft (Bloodgood and Sabatini, 2005; Harnett et al., 2012) but see (Popovic et al., 2014). In this way activation of AMPA receptors may result in sufficient local depolarization to facilitate relief of adjacent NMDA receptors from Mg2+ block. Additionally, AMPA receptors lacking GluA2, which are calcium-permeable, could also contribute to these transients (Hollmann et al., 1991). To examine the role of AMPA receptor activation on mSCTs, we performed the same analysis above in the presence of AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). In these experiments NBQX (5 µM) did not affect mSCT frequency (Figure 4A). This argues against a direct (e.g., via calcium-permeable GluA2 lacking receptors) or indirect (via local depolarization) contribution of AMPA receptors to mSCTs (Figure 4A).

Figure 4 with 2 supplements see all
Spontaneous Ca2+ transient generation is decreased by blocking release of Ca2+ release from internal stores but not by blocking the AMPA receptors, L-type Ca2+ channels or group I mGluRs.

(A) Traces (top) from images taken before and after treatment with the AMPA receptor blocker NBQX show no change in Ca2+ transient frequency (bottom) (p = 0.78 with Student's paired t-test, N = 9 experiments, 2 cultures). (B) Imaging in the presence of the specific L-type calcium channel blocker nimodipine (5 µM) does not affect spontaneous Ca2+ transient frequency (bottom) (p = 0.89 with Student's paired t-test. N = 7 experiments 1 culture). (C) Imaging in the presence of mGluR1 and mGluR5 blockers YM202074 and fenobam produce no differences in spontaneous Ca2+ transient frequency (p = 0.26 via Student's t-test. N = 8 cells, 2 cultures). (D) Application of internal Ca2+ store blocker dantrolene produces a significant drop in mSCT frequency in before/after experiments. (p = 0.008, Student's paired t-test, N = 9 experiments, 3 cultures). (E) Fluo-4 AM example traces and frequency quantification from cells recorded in TTX then in TTX + 30 µM Ryanodine. In before/after imaging experiments, 15 min treatment of the use dependent ER Ca2+ channel blocker ryanodine decreased the frequency of observed Ca2+ transients (p = 0.004, N = 8 via Student's paired t-test). (F) Example traces and frequency quantification of cells pre treated with ryanodine and then imaged first in Tyrode's solution containing no Mg2+ followed by Tyrode's solution containing 1.25 mM Mg2+. Ca2+ transients are measured in Mg2+ free solution but not in 1.25 mM Mg2+. (p = 0.024, via Student's unpaired t-test. N = 7 experiments 2 cultures).

https://doi.org/10.7554/eLife.09262.007

Although experiments presented above showed that the NMDA receptor activity is responsible for triggering the majority of mSCTs in response to spontaneous glutamate release, it remains possible that L-type Ca2+ channels may also contribute this activity as they have been shown to open near resting membrane potentials (Kavalali and Plummer, 1996; Magee et al., 1996). Therefore, we also tested if L-type Ca2+ channel activity contributed to the mSCT activity. In these experiments, treatment with the L-type Ca2+ blocker nimodipine (5 µM) did not significantly affect mSCT frequency (Figure 4B) indicating that these channels do not contribute to the Ca2+ transients. However, here we should note that L-type channel activity may still be involved in setting resting Ca2+ levels and thus impact signaling (Wang et al., 2011). Despite producing no change in mSCT frequency in this assay, nimodipine was able to decrease Ca2+ influx in a separate assay (Figure 4—figure supplement 1).

In subsequent experiments we also tested the potential role of Gq-coupled metabotropic glutamate receptor subtypes 1 and 5 in maintenance of Ca2+ transients. These group I mGluRs can affect Ca2+ signaling via activation of phospholipase C and IP3 generation (Skeberdis et al., 2001; Topolnik et al., 2006). However, application of the mGluR1 antagonist YM202074 and mGluR5 antagonist Fenobam did not cause a significant change in mSCT frequency, indicating that activation of these receptors does not contribute to these Ca2+ transients (Figure 4C). Despite producing no change in the measured mSCT frequency, these drugs were shown to be blocking their target receptors in a separate assay (Figure 4—figure supplement 2).

mSCT generation requires Ca2+ release from internal stores

In hippocampal pyramidal cells, NMDA receptor opening by evoked glutamate release elicits larger Ca2+ transients through a Ca2+-induced Ca2+ release mechanism (Lei et al., 1992; Emptage et al., 1999). In this form of signaling, the small Ca2+ transient produced by the NMDA receptor opening raises internal Ca2+ concentrations near ryanodine receptors (RyRs) on the endoplasmic reticulum high enough to cause their opening at which point a much larger transient is generated. To test whether this mechanism plays a role in the mSCTs we observe, we imaged with Fluo-4 AM in the presence of dantrolene, which is known to reduce the Ca2+ sensitivity of RyR1 and 3 by blocking their interaction with calmodulin (Fruen et al., 1997). Indeed, when compared to the TTX baseline, cells imaged with TTX + dantrolene had a significantly reduced mSCT frequency (Figure 4D). This finding was validated by using ryanodine, which directly blocks all three RyR isoforms in a use-dependent manner (Hawkes et al., 1992; Meissner and el-Hashem, 1992). To facilitate use-dependent block of RyRs by ryanodine, the baseline mSCT frequency was first collected in TTX and then cells were perfused for 13 min with a solution containing 30 µM ryanodine without TTX to maximize RyR opening and ryanodine block. The cells were then perfused with Tyrode's containing TTX + ryanodine for 2 min before continuing the recording. Under these conditions, the application of ryanodine produced a ∼fivefold decrease in mSCT frequency (Figure 4E). In addition, the Ca2+ transients that were detectable after ryanodine treatment were substantially decreased in amplitude suggesting that they are likely to be produced by a subpopulation of RyRs that remained unblocked or incompletely blocked (average TTX ∆F/Fo = 0.052 ± 0.001, TTX + Ryanodine ∆F/Fo = 0.045 ± 0.001, p = 0.002 Student's unpaired t-test, n = 199 events in TTX, 110 events in TTX + ryanodine, 8 experiments, 2 cultures). Treatment with dantrolene or ryanodine is presumed to decrease mSCT frequency by blocking RyRs responsible for producing the Ca2+ transient. With these inhibitors present, further NMDA openings can no longer trigger an mSCT. In fact, the efficacy of ryanodine in this case allowed further investigation of the pure NMDA transient under these experimental conditions. We incubated neurons with ryanodine for 15 min to block RyRs and then loaded them with Fluo-4 AM as before. These cells were imaged in Tyrode's solution containing TTX but no extracellular Mg2+ to allow maximal NMDA currents. Under these conditions Ca2+ transients were observed, but when 1.25 mM Mg2+ was again added no further transients could be measured (Figure 4F). These results illustrate that under physiological concentrations of Mg2+, Fluo-4 cannot detect the NMDA Ca2+ transient without further amplification from Ca2+ induced Ca2+ release.

Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling

In the next set of experiments, we aimed to examine the physiological impact of RyR-dependent mSCTs by focusing on the putative role of these Ca2+ signals in regulation of synaptic efficacy. For this purpose, we investigated the role of mSCTs in homeostatic synaptic scaling, which is a compensatory mechanism where neurons scale the strength of their synaptic inputs multiplicatively in a uniform manner in response to global increases or decreases in activity (Turrigiano et al., 1998). This response involves the synthesis and insertion of new AMPA receptors and can be strongly induced by blocking both action potentials and NMDA receptors (Sutton et al., 2004). Importantly, although synaptic scaling in response to activity blockade occurs within a time frame of 24–48 hr, suppression of resting synaptic activity mediated by spontaneous neurotransmitter release events results in more rapid synaptic scaling detectable within hours (Sutton et al., 2006; Nosyreva et al., 2013). This suggests that NMDA receptor activation at rest maintains synaptic homeostasis. However, the mechanism by which NMDA receptor activity near resting membrane potentials signals to translation machinery, in particular to eEF2 kinase, has been unclear, especially when one considers the relatively small ion conductance of NMDA receptors at rest due to Mg2+ block (Espinosa and Kavalali, 2009).

To investigate the role of RyR-dependent mSCTs in homeostatic synaptic scaling, hippocampal neurons were incubated for 3 hr in culture media containing TTX + vehicle (negative control), TTX + ryanodine, or TTX + AP5 as positive control. Neurons were then perfused with Tyrode's solution and whole cell voltage clamp recordings were made in 1 µM TTX, 50 µM PTX and 50 µM AP5 to isolate AMPA-mEPSCs. Under these conditions, the amplitude distributions of AMPA-mEPSCs obtained from neurons treated previously with TTX + ryanodine as well as those treated with TTX + AP5 showed a significant rightward shift towards larger amplitudes compared to the control condition (Figure 5A,B). When the collected mEPSC amplitudes were plotted rank order in control vs TTX + ryanodine, a linear fit revealed a scaling factor of 1.28 indicating that cell-wide, mEPSC amplitudes increased uniformly 28% over 3 hr with TTX + ryanodine treatment (Figure 5C). This increase in mEPSC amplitudes was not as pronounced as was found with the positive control (TTX + AP5) which may correlate with the finding that ryanodine treatment does not block mSCTs as completely as AP5 (Figures 2D, 4E). It is important to note that while other groups have reported an immediate decrease in mEPSC frequency with the acute application of ryanodine (Emptage et al., 1999), in our system the mEPSC frequencies in neurons treated with ryanodine for 15 min were indistinguishable from those incubated with vehicle as control (TTX mEPSC freq = 7.59 Hz ± 1.75, TTX + Ryanodine mEPSC freq = 8.92 ± 1.13, p = 0.54 using Student's t-test, N = 7 cells from 5 coverslips, 2 cultures). Since the acute application of ryanodine does not alter mEPSC frequency in this system we believe the synaptic scaling effect mainly results from ryanodine acting at the postsynapse to block mSCT activity.

Treating cells with ryanodine + TTX produces a protein synthesis dependent increase in mEPSC frequency and amplitude indicative of homeostatic synaptic scaling.

(A) Example voltage clamp recordings from cells treated with TTX + vehicle (negative control, N = 9 cells from 5 coverslips, 3 cultures), TTX + ryanodine (N = 8 cells from 5 coverslips, 4 cultures) or TTX + AP5 (positive control, N = 6 cells from 4 coverslips, 2 cultures) for 3 hr. (B) Cumulative probability histogram showing significant rightward shifts (increases) in the amplitude of AMPA mEPSCs of cells treated with TTX + ryanodine (red line, p = 1.74 × 10−17, D = 0.151), or TTX + AP5 (p = 8.79 × 10−40, D = 0.255) vs control via Kolmogorov–Smirnov test. (C) Rank order plot of TTX + vehicle mEPSC amplitudes vs TTX + ryanodine showing a multiplicative scaling factor of 1.28. (D) Example voltage clamp recordings from cells pretreated for 30 min with the protein synthesis inhibitor anisomycin and then TTX + vehicle (N = 6 cells from 5 coverslips, 4 cultures) or TTX + ryanodine for 3 hr (N = 7 cells from 5 coverslips, 2 cultures). (E) Cumulative probability histogram of mEPSC amplitudes shows no significant difference between treatment groups when pretreated with anisomycin (p = 0.078, D = 0.052 via Kolmogorov–Smirnov test). (F) Rank order plot of mEPSC amplitudes indicates that anisomycin pretreatment abolishes scaling between treatment groups.

https://doi.org/10.7554/eLife.09262.010

In earlier experiments homeostatic synaptic scaling that occurs after blockade of resting NMDA receptor activity was shown to rely on protein synthesis, in particular synthesis of new AMPARs rather than the insertion of existing ones (Sutton et al., 2006, 2007). In order to test whether this is the case for RyR block-induced synaptic scaling, we repeated the experiment above with neurons that were treated with the protein synthesis inhibitor anisomycin (20 µM) starting 30 min prior to their 3 hr incubation with TTX. Under these conditions, anisomycin completely abolished the increase in AMPA-mEPSC amplitudes as no significant differences were seen in their distribution after TTX + ryanodine treatment compared to treatment with TTX alone (Figure 5D–F).

Previous studies have also shown that a key regulator of protein synthesis, eukaryotic elongation factor 2 (eEF2), is phosphorylated and inactivated by the Ca2+-dependent eEF2 kinase thus blocking protein synthesis under resting conditions (Sutton et al., 2007; Autry et al., 2011; Nosyreva et al., 2013; Gideons et al., 2014). To test whether RyR-mediated mSCTs could be tonically activating eEF2 kinase and thus inhibiting protein synthesis in dendrites, we tested the impact of ryanodine treatment in hippocampal neuronal cultures from eEF2 kinase knockout mice. In hippocampal neurons made from wild-type littermate controls, treating with TTX + ryanodine for 3 hr produced a significant increase in mEPSC amplitudes compared to TTX + vehicle, where plotting the amplitudes in rank order revealed a 42% increase in synaptic strength (Figure 6A–C). When the same experiment was performed using neurons from eEF2 kinase knockout mice, treatment with TTX + ryanodine did not produce a significant shift in mEPSC amplitudes (Figure 6D,E). The rank order plot revealed only a 1% difference in synaptic strength between treatment groups (Figure 6F). Taken together these results suggest that RyR-dependent mSCT-driven signaling acts through Ca2+-dependent eEF2 kinase to maintain synaptic homeostasis.

Ryanodine treatment does not trigger homeostatic synaptic scaling in eEF2 kinase knockout neurons.

(A) Example traces from WT littermate mice with and without 3 hr ryanodine treatment. (B) Cumulative probability histogram shows a significant shift in mEPSC amplitude in TTX + ryanodine treated animals (N = 12 cells, from 7 coverslips, 3 cultures) vs TTX + vehicle control (N = 13 cells from 8 coverslips, 3 cultures) (p = 3.32 × 10−14, D = 0.112 via Kolmogorov–Smirnov test). (C) Rank order plot shows a 1.42 fold increase in synaptic strength after ryanodine treatment. (D) Example traces from eEF2 kinase KO animals with and without ryanodine treatment. (E) Cumulative probability histogram shows no shift in the distribution of mEPSC amplitudes in eEF2K KO animals treated with TTX + ryanodine (N = 9 cells, from 6 coverslips, 3 cultures) vs TTX + vehicle (N = 13 cells from 7 coverslips, 3 cultures) (p = 0.066, D = 0.058 via Kolmogorov–Smirnov test). (F) Rank order plot shows no appreciable multiplicative change in synaptic strength in the eEF2 K KO animals with ryanodine treatment.

https://doi.org/10.7554/eLife.09262.011

Discussion

In this study, we took advantage of multiple Ca2+ indicator probes to examine the properties of Ca2+ transients detected in hippocampal neurons in physiological levels of extracellular Mg2+ in the absence of action potentials. These transients are important because they are key to understanding the Ca2+ signaling events occurring at rest that result in regulation of protein translation and gene transcription leading to synaptic plasticity (Chen et al., 2014; Lalonde et al., 2014) (for review see Kavalali, 2015). Under these conditions we detected robust NMDA receptor dependent Ca2+ transients at a rate of 0.32 ± 0.03 min−1 where previously our group was able to measure a per synapse spontaneous release rate of 0.76 ± 0.03 min−1 by imaging with presynaptic probes (Leitz and Kavalali, 2014). The relatively higher release rate measured earlier may suggest that not every release event is able to generate an mSCT. The link between these Ca2+ transients and spontaneous neurotransmitter release was verified by the parallel increase in mSCT and spontaneous neurotransmitter release frequencies in response to application of hypertonic sucrose. Furthermore, the frequency of Ca2+ transients was also significantly diminished in neurons lacking SNAP-25, which show a substantial reduction in spontaneous release, and in cultures treated with NMDA receptor blockers.

Interestingly GCaMP5K-PSD95, a probe located near the postsynaptic density, revealed only a very small number of events that fulfilled our detection criteria while soluble probes proved to be much better indicators. This observation indicates that although mSCT generation depends on NMDA receptor driven Ca2+ influx, this does not result in strong signals at the postsynaptic density. Rather, mSCTs seem to rely on the activation of Ca2+ release from smooth endoplasmic reticulum which is present in spines or adjacent dendritic regions (Spacek and Harris, 1997).

The generation of mSCTs was not dependent on AMPA receptors, L-type Ca2+ channels or postsynaptic metabotropic glutamate receptor subtypes 1 and 5. While all of these play a role in Ca2+ dynamics under other circumstances the mSCTs we observed under resting conditions were primarily driven by the coupling of the NMDA receptor to internal Ca2+ stores through the ryanodine receptor, as the application of dantrolene or ryanodine produced a marked reduction in both mSCT frequency and amplitude. Earlier studies performed in hippocampal synapses discovered that unitary evoked EPSCs were accompanied by Ca2+ transients that were only minimally dependent on voltage gated Ca2+ channels or AMPA receptors. However, unlike the mSCTs we observe, the application of ryanodine produced only a small reduction in Ca2+ transient amplitude in these experiments (Kovalchuk et al., 2000). This difference may suggest that spontaneous glutamate release-driven Ca2+ transients are more dependent on internal Ca2+ stores compared to Ca2+ transients elicited by evoked release.

In this study, we tested a key prediction of these observations on synaptic plasticity by assessing the role of Ca2+-induced Ca2+ release in synaptic scaling triggered at rest. Our experiments showed that the synaptic scaling produced by the blockade of spontaneous NMDA-mEPSCs is also produced by blocking the Ca2+ release from internal stores indicating a strong link between the two signals. The generation of relatively large store-driven Ca2+ transients provides a critical amplification step for the relatively small NMDA-mEPSCs seen under physiological conditions (Espinosa and Kavalali, 2009; Gideons et al., 2014). The resulting signal is delocalized and pulsatile which may allow synaptic NMDA receptors to exert signaling influence in the surrounding dendritic regions. This could be critical for local translational control as eEF2 localizes to the dendritic shaft rather than dendritic spines (Asaki et al., 2003). The low frequency of observed mSCTs may also be a defining attribute, as the ubiquitous Ca2+ binding protein calmodulin is predicted to interact with different target kinases and enzymes based on the frequency and duration of its activation by free Ca2+ (Saucerman and Bers, 2008; Slavov et al., 2013). Taken together these findings identify a critical missing mechanistic link between spontaneous neurotransmission and the control of dendritic signaling events that regulate synaptic efficacy.

Materials and methods

Cell culture

Hippocampal cultures from Sprague–Dawley rats or eEF2 kinase knockout mice and their wild-type littermate controls were generated from postnatal day 1–3 male and female pups and plated on Matrigel (Corning Inc, NY) coated coverslips as described previously (Kavalali et al., 1999). Neurons were infected with lentivirus at 4 days in vitro. Neurons were used for experiments between 14 to 18 days in vitro.

Dissociated hippocampal cultures from SNAP25 knockout mice and their wild-type littermates were generated from E17-20 embryos and were plated on poly-d-lysine coated coverslips as described previously (Bronk et al., 2007). Neurons were used for experiments 14–18 days in vitro.

Whole cell voltage clamp recordings

Dissociated hippocampal cultures aged 14–18 days in vitro were voltage clamped at −70 mV using an Axon Instruments Axopatch 200B amplifier with access resistances less than 25 MΩ for each recording. Internal pipette solution contained (in mM): 120 K-Gluconate, 20 KCl, 10 NaCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP and 0.3 Na-GTP at pH 7.3. To isolate AMPA-mEPSCs, the extracellular solution contained 1 µM TTX, 50 µM picrotoxin (PTX, to block mIPSCs) and 50 µM (2R)-amino-5-phosphonovaleric acid (AP5), 2 mM Ca2+ and 1.25 mM Mg2+. All whole cell patch clamp recordings were performed under continuous perfusion. Cells were perfused for 3-min prior to recording to achieve stable baselines. No more than 2 recordings were obtained per coverslip.

Whole cell voltage clamp statistics and analysis

AMPA-mEPSCs were quantified using Synaptosoft MiniAnalysis software. Frequency data was collected by quantifying 4 min per cell starting at the beginning of each recording. To ensure that high frequency cells did not skew the amplitude comparisons by being over represented, 200 mEPSC amplitudes were randomly selected from each recording to build the cumulative probability histograms and rank order plots. Kolmogorov–Smirnov test was performed using Past 3.02 (http://folk.uio.no/ohammer/past/).

Ca2+ imaging

Fluo-4 AM imaging

Neurons were incubated for 10 min in culture media containing 5.6 µM Fluo-4 AM (Life Technologies, Grand Island NY). Coverslips were then removed and washed for 2 min prior to recording with Tyrode's solution containing (in mM) 150 NaCl, 4 KCl, 1.25 MgCl2, 2 CaCl2 and 10 TES buffer, pH adjusted to 7.4. Where solutions are noted to have 0 mM Ca+ the solution is prepared from deionized water with no added Ca2+. All solution changes except +100 mM hypertonic sucrose include 2 min of wash time in the new solution to ensure full application of the new conditions. Neurons were imaged using a 40× objective on a Nikon TE2000-U microscope. Images were collected at 10 frames per second using an Andor xION Ultra EMCCD camera for a duration of ∼ 2 min. Event frequencies per ROI were estimated using the population average obtained from 72 ROIs monitored per experiment. Our analysis, therefore, refers only to average per ROI frequency per experiment. Illumination was provided by a Sutter DG-4 arc lamp using a 470 ± 40 nm bandpass excitation filter. Post experiment synapse visualization used a 548 ± 10 nm filter to excite Syb2-mO and Tyrode's solution containing 50 mM NH4Cl to maximize fluorescence. The emission filter in place allowed 515 ± 15 nm and 590 ± 20 nm bands to pass. Fluo-4 traces were generated by measuring circular ROI, 3 µm radius centered over Syb2-mO puncta.

Single cell Fluo-4 imaging

Uninfected neurons were loaded with indicator by using the whole cell voltage clamp configuration described above. The patch pipette contained 200 µM Fluo-4 pentapotassium salt (Life Technologies, Grand Island NY).

Soluble GCaMP5K imaging

Wild type neurons expressing Syb2-mO were transfected with pFU-GCaMP5K using lipofectamine 3000 (Life Technologies, Grand Island NY) 8 hr prior to imaging. Images were collected as above.

GCaMP5K-PSD95 imaging

Neurons expressing GCaMP5K-PSD95 via lentiviral infection were imaged as indicated above except using a 100× objective. Images were collected at 8 frames per second to minimize single frame noise.

Ca2+ transient analysis and statistics

Ca2+ transient frequency was derived from imaging traces by counting Ca2+ transients where the signal peak had a 2-point slope greater than 70 A.U. (350 units/s over a 200 ms window) and amplitude greater than 0.035 ∆F/Fo. Ca2+ signals were not counted if their peak width was greater than 5 s. Maximum peak amplitude was required to be 2 standard deviations greater than the mean of the signal in the previous 2 s. Single high points were not counted. Detected peaks were ignored if another peak was detected in the following 400 ms to prevent the double counting of slower mSCTs. All error bars represent standard error of the mean except in Figure 1A–D where standard deviation is used. Rise and decay times are displayed as τ, where τ is the time in seconds necessary to reach (11e)ΔF for the rising phase or (1e)ΔF for the decay phase based on a single exponential fit line obtained with Axon Clampfit 9.0.1.07. All statistical tests were performed using Graphpad Prism 6.01.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
    Spontaneous subthreshold activity at motor nerve endings
    1. P Fatt
    2. B Katz
    (1952)
    The Journal of Physiology 117:109–128.
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
    [3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia
    1. MJ Hawkes
    2. TE Nelson
    3. SL Hamilton
    (1992)
    The Journal of Biological Chemistry 267:6702–6709.
  17. 17
  18. 18
    Voltage dependence of NMDA-activated macroscopic conductances predicted by single-channel kinetics
    1. CE Jahr
    2. CF Stevens
    (1990)
    The Journal of Neuroscience 10:3178–3182.
  19. 19
  20. 20
  21. 21
    Multiple voltage-dependent mechanisms potentiate calcium channel activity in hippocampal neurons
    1. ET Kavalali
    2. MR Plummer
    (1996)
    The Journal of Neuroscience 16:1072–1082.
  22. 22
    NMDA receptor-mediated subthreshold Ca(2+) signals in spines of hippocampal neurons
    1. Y Kovalchuk
    2. J Eilers
    3. J Lisman
    4. A Konnerth
    (2000)
    The Journal of Neuroscience 20:1791–1799.
  23. 23
  24. 24
  25. 25
  26. 26
    Activation of silent synapses by rapid activity-dependent synaptic recruitment of AMPA receptors
    1. D Liao
    2. RH Scannevin
    3. R Huganir
    (2001)
    The Journal of Neuroscience 21:6008–6017.
  27. 27
    Dihydropyridine-sensitive, voltage-gated Ca2+ channels contribute to the resting intracellular Ca2+ concentration of hippocampal CA1 pyramidal neurons
    1. JC Magee
    2. RB Avery
    3. BR Christie
    4. D Johnston
    (1996)
    Journal of Neurophysiology 76:3460–3470.
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
    Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat
    1. J Spacek
    2. KM Harris
    (1997)
    The Journal of Neuroscience 17:190–203.
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44

Decision letter

  1. Indira M Raman
    Reviewing Editor; Northwestern University, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled “Spontaneous neurotransmission signals through store-driven Ca2+ transients to maintain synaptic homeostasis” for further consideration at eLife. Your article has been favorably evaluated by Eve Marder (Senior editor), a Reviewing editor, and three reviewers. The manuscript has been greatly improved over your previous submission but there are two minor corrections that must be addressed before acceptance, regarding apparent inconsistencies/ambiguities in the text. The first may be a typo regarding AP5 sensitivity reported in text vs. legend, and the second has to do with quantification of a low rate from relatively short recordings (see details below).

In general the reviewers were convinced by the revisions. Two reviewers said: “I am satisfied with all of the revisions and hope to see the paper published” and “The authors have addressed all my concerns reasonably. I have no further concerns.”

The third writes: “Most of my concerns were satisfactorily addressed but two points remain.” That reviewer listed the two inconsistencies:

1) There is an apparent contradiction between the text in subsection “mSCTs are driven by spontaneous neurotransmitter release” and in the legend to Figure 3. Text: “Neurons derived from SNAP25 knock out animals had a significantly decreased baseline mSCT frequency. Also, these transients remained sensitive to AP5, which is again consistent with mSCT generation being driven by spontaneous vesicle release (Figure 3C, D).”

The statement “these transients remained sensitive to AP5” seems to be contradicted by the statement in the legend: “The remaining mSCTs in the KO animals are unaffected by AP5.” Is “unaffected” incorrect in the legend? This needs to be clarified in the manuscript.

2) The recordings were 2 minutes long. In the third paragraph of the subsection “Visualization of miniature spontaneous Ca2+ transients in hippocampal neurons” it states an mSCT frequency of 0.009 per min per ROI. It needs to be explained how such a low frequency (∼0.5 per hour) can be obtained from 2 min long recordings.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled “Spontaneous neurotransmission signals through store-driven Ca2+ transients to maintain synaptic homeostasis” for consideration at eLife. Your full submission has been evaluated by Eve Marder (Senior editor), a Reviewing editor, and three peer reviewers, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work cannot be accepted in its present form for publication in eLife. The reviewers made specific suggestions for improvement of the manuscript, however, and if you wish to do the necessary experiments and carry out the suggested revisions, we would be willing to consider a new version of the manuscript. This decision was made not because we didn't find the work potentially exciting, but because the experiments that the reviewers thought important might take more than several weeks to accomplish, and it is against eLife policy to ask for extensive new experiments for a revision.

In the discussion of the reviews, three major topics emerged, which all reviewers generally agreed upon. The first is the need for a better description of the analysis, which will likely require some modification of the analysis. Ideally these would resolve the inconsistencies noted in the first review. The second has to do with the N-values and the validity of the statistics. Not only were N-values low, but explicit indication of what “N” refers to in the experiments would also be appropriate (how many independent cultures, how many different coverslips, whether similar #'s of mini's collected and considered from each cell, etc). Third were experimental questions about results that appear contradictory (why ryanodine affects Ca transient frequency but only affects mEPSC amplitude) or ambiguous (in Figure 1F, what is the relative sensitivity of the two different Ca2+ indicators, and why it is valid to directly compare data obtained using those different constructs).

The complete reviews are included below to facilitate a new submission, should you decide to submit one. Please note that it would likely require some additional experiments to address the concerns of low N as well as rewriting and reanalysis.

Reviewer #1:

In this manuscript the author present experiments investigating miniature spontaneous Ca2+ transients (mSCTs) in hippocampal neurons. They report that these are activated by spontaneous glutamate release events and enhanced by Ca2+-induced Ca2+release, confirming previous results. New insight comes mainly from Figures 5 and 6. The authors show that inhibition of Ca2+-induced Ca2+ release increases mEPSC amplitudes, indicative of synaptic scaling. This increase is abolished by a protein synthesis inhibitor and in neurons form eEF2 kinase knock-out mice, indicating that ryanodine induced synaptic scaling acts via eEF2 kinase activation.

While there is some interesting new insight from this manuscript, much of it largely confirms previous results, several sections are very poorly written and a number of technical questions arise. In particular, the description of mSCT detection and analysis is inconsistent, raising serious questions regarding the significance of observed phenomena.

Specific points:

1) The event detection/identification needs clarification (Discussion and second paragraph, Figure 2). For the 2 SD threshold 5% of points will exceed the threshold. The Methods section gives only criteria of maximum duration. Was there a minimum duration?

2) How were rise times and decay times defined and determined?

3) The average/median amplitudes of ∆F/F=0.054/0.048 seem to be nearly equal to 2 SD in Figure 2A.

4) The legend of Figure 2 states that events having “peak ∆F/F greater than 0.04 were counted if the peak fluorescence value was 2 standard deviations greater than the mean fluorescence”. Methods, subsection “CA2+ transient analysis and statistics” states 0.035, and the distribution in Figure 2B (right) shows counts for amplitudes even smaller. The analysis seems inconsistent.

5) According to the Methods section Fluo-4 and GCaMP5k were excited with 470± 40 nm filter, which will also excite Syb2-mOrange, which will change it fluorescence upon vesicle fusion. What emission filter was used to exclude crosstalk?

6) For Figure 1 ROIs were selected over Syb2mOrange puncta determined after NH4Cl addition. What was the size and shape of the ROIs?

7) Introducing Fluo-4 salt with a patch pipette reduced observation frequency of mSCTs and the authors interpret this as being due to wash-out. However, it seems that the pipette solution contained high gluconate, which is known to buffer Ca2+ (Kd∼35 mM). Although Kd may appear high it would be expected to affect [Ca2+] due to its very high concentration. Washout should produce a decrease in frequency over time compared to Fluo-4 AM, was this observed?

8) Frequency of mSCTs measured with GCaMP5k-PSD95 was ∼1 very 2 hours. How long were the recordings?

9) In the fourth paragraph of the subsection “Visualization of miniature spontaneous CA2+ transients in hippocampal neurons” and in Figure 1F: The authors claim that differences were insignificant except comparing GCaMP5k-PSD to Fluo-4 AM. From the error bars in Figure 1F this seems not to be supported by the data.

10) Does the lack of events with GCaMP5k-PSD indicate that these are extrasynaptic? If so, this should be stated more clearly.

11) Figure 4A-C: For these negative results – how did the authors ensure that the blockers actually worked in these experiments effectively blocking the respective receptors and L-type Ca current?

12) The legend of Figure 4: “The remaining mSCTs in the KO animals are unaffected by AP-5”. This seems not to refer to the data shown in Figure 4D.

13) Figure 5B shows a much larger effect of AP5 than ryanodine. How do the authors interpret this result?

Reviewer #2:

This is an interesting and timely paper on a topic that has received much recent attention in neuroscience. I believe that this paper should be accepted, but there is one somewhat large issue regarding sample size that need be addressed first:

Throughout the paper, the sample size for the experiments is low (N of 5 to 6 is not uncommon). For example, in Figure 4, which encompasses five separate experiments, the total N for all five experiments appears to be less than 30.

(This general issue, RE sample size and statistics in neuroscience, is discussed here: http://www.nature.com/nrn/journal/v14/n5/full/nrn3475.html). It seems that the N should be increased in some experiments reported here.

In the first paragraph of the subsection “Visualization of miniature spontaneous CA2+ transients in hippocampal neurons”: The frequency of spontaneous Ca2+ transients was 0.32 per minute. If you sum the Ca2+ transients across all release sites in entire cell, do you get a Ca2+ transient frequency that roughly matches the mEPSC frequency (∼1Hz)? If so, that would be a good validation of the method. If not, then either there is a problem with the method or there's some fraction of release that does not trigger NMDAR/CICR signaling, which would be good to know. If this is not technically feasible, please explain.

Figure 5: It is surprising that ryanodine had no effect on mEPSC frequency. From the representative traces, it would appear that ryanodine did have an effect. Maybe these are not very representative? Or, do the authors think there was a slight effect of ryanodine in mEPSC frequency that did not reach significance? Please address.

Reviewer #3:

In this manuscript Reese and Kavalali report how spontaneous neurotransmission influences synaptic homeostasis. They show action potential independent calcium transients in the dendritic region using multiple calcium indicators. The calcium transients required NMDA receptor activity and calcium release from internal stores. Furthermore they show that reducing the calcium transient through inhibiting NMDA receptors or blocking internal stores results in synaptic scaling mediated by protein synthesis and eukaryotic elongation factor 2 (eEF2). The authors suggest that spontaneous neurotransmission through NMDA receptor driven calcium transient is required for synaptic homeostasis.

The manuscript has several interesting findings, however one major issue is that there is quite a jump between the story on what is the source of spontaneous dendritic calcium transients to how it affects the synapse. Either they need to provide better transition or posit the paper in a different manner. Figures 1 through 4 make sense together, but the last two figures, which are addressing synaptic scaling, appear without much of a transition.

1) In Figure 1, the calcium transient from Fluo-4 AM is not increased in 0 magnesium, the authors do point that out. In light of what is indicated in the Introduction, where they state '…that calcium signal is 20% of the full calcium signal of unblocked NMDA…', it is quite surprising that the signal is not higher in 0 magnesium. This needs to be addressed. Furthermore, because Fluo-4 AM detects calcium events both in the presynapse and postsynapse, an explanation or experiment is required to distinguish these two sources. How did they define the calcium events as dendritic only? Figure 1B, is the impermeable Fluo-4 experiment conducted in Syb2-mO infected neurons? If so, it is worth checking if there is any spontaneous transients in the presynaptic terminal of cells that express Syb2-mO.

2) The authors showed mSCT frequency is nicely correlated with mEPSC frequency, when influenced by presynaptic players (Figure3). On the other hand intracellular calcium store blockers, Dantrolene and Ryanodine potentially only affecting the dendrite each reduce mSCT frequency (Figure 4). It is surprising and also hard to understand how calcium release from internal stores affects frequency rather than amplitude or kinetics of the calcium transients. It would be important to address if the presynapse is affected by Dantrolene and Ryanodine.

3) Figure 5, the authors indicate that ryanodine and AP5 each only influence mEPSC amplitudes not the frequency. From the sample trace it appears that frequency is actually increased in both cases, a more representative trace is needed here.

4) Why does ryanodine affect the calcium transient frequency, but affects only the mEPSC amplitude? This seems contradictory. I would expect an effect on the mEPSC frequency as well.

https://doi.org/10.7554/eLife.09262.012

Author response

1) There is an apparent contradiction between the text in subsection “mSCTs are driven by spontaneous neurotransmitter release” and the legend to Figure 3. Text: “Neurons derived from SNAP25 knock out animals had a significantly decreased baseline mSCT frequency. Also, these transients remained sensitive to AP5, which is again consistent with mSCT generation being driven by spontaneous vesicle release (Figure 3C, D).

The statementthese transients remained sensitive to AP5seems to be contradicted by the statement in the legend:The remaining mSCTs in the KO animals are unaffected by AP5.Isunaffectedincorrect in the legend? This needs to be clarified in the manuscript.

What is meant is that the overall population of mSCTs in the knockout animals is sensitive to AP5, but after AP5 treatment there are some mSCTs that remain unaffected. The legend for Figure 3D has been edited and now reads:

D. Fluo-4 imaging in cultures made from SNAP25 KO and littermate control mice reveal that the KO cultures have a substantially decreased mSCT frequency. In this setting, AP5 treatment greatly decreases but does not completely abolish the remaining mSCTs. (WT, TTX vs WT, TTX+AP5 p = 0.010. WT, TTX vs KO, TTX p = 0.008. KO TTX vs KO TTX+AP5 p = 0.003, via 1-way ANOVA with Tukey’s multiple comparisons, N=8 experiments in WT cells and 9 experiments in KO cells from 3 cultures).

2) The recordings were 2 minutes long. In the third paragraph of the subsection “Visualization of miniature spontaneous Ca2+ transients in hippocampal neurons” it states an mSCT frequency of 0.009 per min per ROI. It needs to be explained how such a low frequency (∼0.5 per hour) can be obtained from 2 min long recordings.

Recordings were 2 minutes long to prevent photodamage and bleaching, which would have resulted in less reliable data. However, in each experiment we measured around 72 ROIs and estimated event frequencies per ROI using the population average (equivalent to ∼ 2 hrs per experiment). In fact, for this reason we do not report per synapse frequency distributions in the manuscript and only refer to average per ROI frequency per experiment. This point is now clarified in the Methods section:

Event frequencies per ROI were estimated using the population average obtained from 72 ROIs monitored per experiment.”

[Editors’ note: the author responses to the previous round of peer review follow.]

In the discussion of the reviews, three major topics emerged, which all reviewers generally agreed upon. The first is the need for a better description of the analysis, which will likely require some modification of the analysis. Ideally these would resolve the inconsistencies noted in the first review.

We followed the reviewers’ advice and now expanded the description of our analysis.

The second has to do with the N-values and the validity of the statistics. Not only were N-values low, but explicit indication of whatNrefers to in the experiments would also be appropriate (how many independent cultures, how many different coverslips, whether similar #'s of mini's collected and considered from each cell, etc).

N-values are now clearly stated in each figure legend as number of coverslips or cells and the number of cultures. In several cases we conducted new experiments to increase N-values.

Third were experimental questions about results that appear contradictory (why ryanodine affects Ca transient frequency but only affects mEPSC amplitude) or ambiguous (in Figure 1F, what is the relative sensitivity of the two different Ca2+ indicators, and why it is valid to directly compare data obtained using those different constructs).

We now clarified these issues in the manuscript. Briefly, mEPSCs report synaptic AMPA receptor activity, whereas Ca2+ transients reflect RyR mediated Ca2+ release events triggered by residual NMDA receptor activity (seen in the presence of Mg2+). Therefore, the two measures, although both dependent on spontaneous glutamate release and occur concurrently, report distinct nodes in the signaling cascade. Please also note that in our results Ca2+ transients are not sensitive to AMPA blockers in contrast to their extreme sensitivity to NMDA receptor block (Figure 2D).

Overall, in revising this manuscript, we have done the following:

1) We have clarified the description for the signal processing and analysis of Fluo-4 AM calcium transients as well as verifying that all measurements included in this manuscript conform to these analysis standards. We have included in this clarification a more detailed Figure 2A that illustrates the process used in detecting these calcium signals.

2) We have performed new measurements to increase N numbers throughout. New experiments have been added to the experiments in Figure 2C so that the final N number is 8.

3) New experiments have been performed to add to Figures 3A and 3B so that the final N numbers are 8 and 9 respectively. This new data increases the confidence of our findings by further decreasing the p values to 0.002 and 0.028 respectively.

4) New experiments have been performed to add to panels 4C, 4D and 4E. These experiments bolster the N numbers to 8, 9 and 8 respectively.

A new experiment has been performed to clarify the makeup of the calcium transients detected with Fluo-4 AM. This experiment is now displayed as Figure 4F. Briefly, ryanodine treated cells (ER store calcium is blocked) were recorded in Fluo-4 AM with and without magnesium in order to confer or relieve the voltage dependence of the NMDA receptor. With no magnesium in the bath the undamped calcium transient of the NMDA receptor can be measured but when magnesium is added to the bath solution no further transients can be measured. This experiment illustrates that the calcium transients measured in 1.25 mM Mg2+ with Fluo-4 AM include a negligible amount of signal from the NMDA receptor itself and are primarily driven by ER stores.

6) A control experiment has been done to illustrate that under these recording conditions, no appreciable signal from the red channel (Synaptobrevin2-mOrange) is bleeding into the green channel (Fluo-4 AM). Under the conditions used throughout the paper to detect calcium transients with Fluo-4 AM, no appreciable bleed through was detected between the two channels.

7) Control experiments were performed to confirm the efficacy of Nimodipine. The same stock solution that was previously used in the manuscript was again tested and found to be working in a separate assay.

Control experiments were performed to confirm the efficacy of YM202074 and Fenobam. The same stock solutions that were previously used in the manuscript was again tested and confirmed to be working in a separate assay.

Reviewer #1:

In this manuscript the author present experiments investigating miniature spontaneous Ca2+ transients (mSCTs) in hippocampal neurons. They report that these are activated by spontaneous glutamate release events and enhanced by Ca2+-induced Ca2+release, confirming previous results. New insight comes mainly from Figures 5 and 6. The authors show that inhibition of Ca2+-induced Ca2+ release increases mEPSC amplitudes, indicative of synaptic scaling. This increase is abolished by a protein synthesis inhibitor and in neurons form eEF2 kinase knock-out mice, indicating that ryanodine induced synaptic scaling acts via eEF2 kinase activation.

While there is some interesting new insight from this manuscript, much of it largely confirms previous results, several sections are very poorly written and a number of technical questions arise. In particular, the description of mSCT detection and analysis is inconsistent, raising serious questions regarding the significance of observed phenomena.

We thank the reviewer for his/her constructive criticisms. We respectfully disagree that our findings are largely confirmatory. We believe that our results provide insight into a critical open question in neuroscience, namely how spontaneous glutamate release events manage to regulate neuronal signaling and synaptic efficacy. We addressed the technical issues raised by the reviewer as follows:

Specific points:

1) Discussion, second paragraph, Figure 2: The event detection/identification needs clarification. For the 2 SD threshold 5% of points will exceed the threshold. The Methods section gives only criteria of maximum duration. Was there a minimum duration?

Detail has been added to the Methods section. The minimum duration for an event was 200 ms as single high points were not counted. However, very few detected events were this fast as you can see from the decay times shown in Figure 2B.

2) How were rise times and decay times defined and determined?

The Materials and methods as well as the Figure 2 legend have been updated.

Briefly, the decay time (τ) is the time that it takes the signal to decrease to 1/e times the peak value (∼37% of the peak). The rise time (τ) is the time that it takes for the signal to reach 1/1-e times the peak value from baseline (∼63% of the max value).

3) The average/median amplitudes of ∆F/F=0.054/0.048 seem to be nearly equal to 2 SD in Figure 2A.

We now better illustrate our selection criteria in Figure 2A. The analysis requires that when the two point slope exceeds 70 (350 A.U./sec) and the ∆F/Fₒ of the second point exceeds 0.035, the amplitude of the second point must exceed 2 SD greater than the mean of the signal 2 seconds previous to be counted as a peak. In Figure 2A, the ∆F/Fₒ threshold is represented by the red line and the gray shaded region shows the area covered by 2SD +/-the mean. For comparison, the bottom red trace shows the two point slope (first derivative approximation) and the 350 A.U/sec threshold.

4) The legend of Figure 2 states that events havingpeak ∆F/F greater than 0.04 were counted if the peak fluorescence value was 2 standard deviations greater than the mean fluorescence. Methods, subsection “CA2+ transient analysis and statistics” states 0.035, and the distribution in Figure 2B (right) shows counts for amplitudes even smaller. The analysis seems inconsistent.

We apologize for this oversight. We now corrected this error. The actual value we used in our analysis is 0.035 (not 0.04). Figure 2B has been re-analyzed and now includes amplitude data taken directly from the analysis files.

5) According to the Methods section Fluo-4 and GCaMP5k were excited with 470± 40 nm filter, which will also excite Syb2-mOrange, which will change it fluorescence upon vesicle fusion. What emission filter was used to exclude crosstalk?

We thank the reviewer for pointing out this important issue. We have two answers to this question:

A) A dichroic filter is in place that allows both wavelengths to pass. This information is now included in Methods. The dichroic emission filter allows 515 ± 15 nm and 590 ± 20 nm bands to pass. This configuration allows the emission wavelengths for both mOrange and Fluo-4 to pass.

In our hands, synaptobrevin-mOrange (unlike synaptophysin-pHtomato or vGlut-pHluorin see Leitz and Kavalali, 2014) is not suited to single vesicle imaging due to its poor signal to noise ratio and the rapid lateral diffusion of the synaptobrevin probe. To illustrate this, dual channel imaging was performed alternating between Fluo-4 AM and Syb2-mO excitation. Images were collected at the same exposure and gain settings as all other Fluo-4 AM experiments (100 ms exposure). 382 peaks were detected in the fluo-4 channel and aligned by peak value to generate an average event. The corresponding Syb2-mO signal was also averaged, but no signal is detected (see Figure 2–figure supplement 1).

6) For Figure 1 ROIs were selected over Syb2mOrange puncta determined after NH4Cl addition. What was the size and shape of the ROIs?

ROIs were in 3 µm radius around the syb2-mOrange puncta. We now included this information in the Methods section. The need for larger ROIs was necessitated by the fact that mSCTs (seen in the presence of Mg2+) typically occur within the vicinity but not directly juxtaposed to presynaptic terminals. The frequency of the mSCTs and their sensitivity to AP5 and alterations in spontaneous release supports their synaptic origin.

7) Introducing Fluo-4 salt with a patch pipette reduced observation frequency of mSCTs and the authors interpret this as being due to wash-out. However, it seems that the pipette solution contained high gluconate, which is known to buffer Ca2+ (Kd ∼35 mM). Although Kd may appear high it would be expected to affect [Ca2+] due to its very high concentration. Washout should produce a decrease in frequency over time compared to Fluo-4 AM, was this observed?

We were concerned about wash out as well as photobleaching and therefore we limited our acquisition time to 2 min. This duration was also necessitated by the relatively high temporal resolution of our optical recordings.

Author response image 1 shows that over the course of 4 experiments with Fluo-4 pentapotassium salt, we only detect a minor trend towards a decrease in frequency over the recording window.

8) Frequency of mSCTs measured with GCaMP5k-PSD95 was ∼1 very 2 hours. How long were the recordings?

These recordings were ∼2 minutes long due to their relatively high temporal resolution (10 frames per second) as well as due to the concerns indicated above.

9) In the fourth paragraph of the subsection “Visualization of miniature spontaneous CA2+ transients in hippocampal neurons” and in Figure 1F: The authors claim that differences were insignificant except comparing GCaMP5k-PSD to Fluo-4 AM. From the error bars in Figure 1F this seems not to be supported by the data.

We agree with the reviewer that at a glance these data should be statistically significant. However, when we use the appropriate Holm-Sidak multiple comparison test, we only detect a significant difference between GCaMP5k-PSD95 and the Fluo-4 AM conditions. If we use pairwise t-tests, indeed these data all seem significant but we do not think this is appropriate way to compare. All experiments had relatively high numbers except for the impermeable Fluo4 injection out of 15 experiments only 4 yielded interpretable findings.

10) Does the lack of events with GCaMP5k-PSD indicate that these are extrasynaptic? If so, this should be stated more clearly.

GCaMP5K-PSD probe can detect synaptic Ca2+ transients in the absence of Mg2+ (Leitz and Kavalali, 2014 Figure 2 therein). While the lower detection efficiency of GCaMP5K-PSD95 compared to soluble probes does not prove the location of these transients in itself, it does suggest that the bulk of the Ca2+ transients occur in a cellular compartment where it cannot be measured by the postsynaptically localized probe. We have added clarification in the last paragraph of the subsection “Visualization of miniature spontaneous Ca2+ transients in hippocampal neurons”.

In addition, the majority of Ca2+ transients measured with Fluo-4 AM are sensitive to ryanodine block which indicates that the ER is the bulk source of the Ca2+ ions for these events.

11) Figure 4A-C: For these negative results – how did the authors ensure that the blockers actually worked in these experiments effectively blocking the respective receptors and L-type Ca current?

NBQX is a common laboratory drug and is used concurrently in many studies in our lab. Its efficacy has never been problematic (e.g. Gideons et al., 2014, PNAS). Nimodipine, YM202074 and Fenobam were obtained for this project alone so we tested the efficacy of these compounds as follows: Nimodipine, the L-Type Ca2+ blocker was tested in our dissociated culture system by loading cells with Fluo-4 AM as elsewhere in the manuscript and in Tyrode’s solution containing 50 µM AP-5 and 20 µM NBQX. In the presence of sustained stimulation to evoke 10 action potentials at 0.2 Hz, the extracellular solution was changed with one that also contained 10 µM Nimodipine and 10 more action potentials were evoked. Our results show that there is a significant decrease in action potential dependent Ca2+ influx after the addition of Nimodipine.

To test whether the mGluR1/5 blockers YM202074 and Fenobam were functioning properly, we employed a similar setup using dissociated cultures loaded with Fluo-4 AM. Transient Ca2+ increases were evoked by perfusing the cells with 100 µM of the mGluR1/5 agonist (S) -3,5-Dihydroxyphenylglycine (DHPG). To test the efficacy of our blockers, baseline fluorescence was collected in solution containing YM202074 and Fenobam and then DHPG was added. During this experiment YM202074 and Fenobam were able to completely block the transient Ca2+ rise associated with DHPG (see Figure 4–figure supplements 1 and 2).

12) The legend of Figure 4:The remaining mSCTs in the KO animals are unaffected by AP-5. This seems not to refer to the data shown in Figure 4D.

“The remaining mSCTs in the KO animals are unaffected by AP5” is in the body of the Figure 3 legend. This sentence is in reference to the fact that the addition of AP- 5 does not decrease the mSCT frequency in the SNAP25 KO animals to zero but rather to very low levels. The remaining mSCTs, though few, persist even in the presence of AP-5.

13) Figure 5B shows a much larger effect of AP5 than ryanodine. How do the authors interpret this result?

We have added the following to the text (subsection “Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling):

“This increase in mEPSC amplitudes was not as pronounced as was found with the positive control (TTX + AP5) which may correlate with the finding that ryanodine treatment (presumably due to its use-dependent nature) does not block mSCTs as completely as AP5 (Figures 2D, 4E).”

Reviewer #2:

This is an interesting and timely paper on a topic that has received much recent attention in neuroscience. I believe that this paper should be accepted, but there is one somewhat large issue regarding sample size that need be addressed first:

Throughout the paper, the sample size for the experiments is low (N of 5 to 6 is not uncommon). For example, in Figure 4, which encompasses five separate experiments, the total N for all five experiments appears to be less than 30.

(This general issue, RE sample size and statistics in neuroscience, is discussed here: http://www.nature.com/nrn/journal/v14/n5/full/nrn3475.html). It seems that the N should be increased in some experiments reported here.

We thank the reviewer for pointing these issues out. We now followed the reviewer’s advice and increased the N for the experiments presented in Figures 2D, 3A, 3B, 4C, 4D, 4E (with the lowest N number of 7 for imaging experiments).

It is also worthwhile to note that from Figure 2 on, all of the imaging experiments are done in a before/after format which greatly decreases false findings and confounds by allowing us to contain comparisons within the same cell and use a paired t-test.

In the first paragraph of the subsection “Visualization of miniature spontaneous CA2+ transients in hippocampal neurons”: The frequency of spontaneous Ca2+ transients was 0.32 per minute. If you sum the Ca2+ transients across all release sites in entire cell, do you get a Ca2+ transient frequency that roughly matches the mEPSC frequency (∼1Hz)? If so, that would be a good validation of the method. If not, then either there is a problem with the method or there's some fraction of release that does not trigger NMDAR/CICR signaling, which would be good to know. If this is not technically feasible, please explain.

Spontaneous release rate per synapse has been estimated to be in the order of 1 vesicle per 60-90 seconds (Geppert et al., 1994 Cell; Murthy and Stevens, 1999 Nature Neuroscience, Sara et al., 2005 Neuron). Our recent best measurement of per synapse spontaneous release frequencies comes from a recent publication titled “Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles” (Leitz and Kavalali, 2014 eLife) where presynaptic vGlut-pHluorin imaging yielded an average frequency of 0.76 ± 0.03 release events/synapse/minute. The relatively higher rate per synapse does in this case suggest that not every mEPSC is able to generate an mSCT. This rate comparison has been added to the Discussion.

Figure 5: It is surprising that ryanodine had no effect on mEPSC frequency. From the representative traces, it would appear that ryanodine did have an effect. Maybe these are not very representative? Or, do the authors think there was a slight effect of ryanodine in mEPSC frequency that did not reach significance? Please address.

The acute treatment of neurons with ryanodine did not alter mEPSC frequency. We have included this data to help illustrate that the primary effect of ryanodine in driving mEPSC scaling is the postsynaptic block of mSCT activity rather than driving a change in release frequency. This point has now been clarified in the Results section discussing Figure 5 (subsection “Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling”, second paragraph). Upon close inspection, the example trace shown in Figure 6A was not a good representative and has been replaced.

Reviewer #3:

[…] The manuscript has several interesting findings, however one major issue is that there is quite a jump between the story on what is the source of spontaneous dendritic calcium transients to how it affects the synapse. Either they need to provide better transition or posit the paper in a different manner. Figures 1 through 4 make sense together, but the last two figures, which are addressing synaptic scaling, appear without much of a transition.

We thank the reviewer for this comment. We now revised the text to include a better transition and rationale for the homeostatic synaptic scaling experiments (subsection “Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling”, first paragraph).

1) In Figure 1, the calcium transient from Fluo-4 AM is not increased in 0 magnesium, the authors do point that out. In light of what is indicated in the Introduction, where they state '…that calcium signal is 20% of the full calcium signal of unblocked NMDA…', it is quite surprising that the signal is not higher in 0 magnesium. This needs to be addressed.

It is indeed true that the addition of physiological concentrations of Mg2+ to the extracellular solution decreases the Ca2+ flux through the NMDA receptor significantly. The fact that removing Mg2+ from the extracellular solution did not yield larger mSCT amplitudes as measured by Fluo-4 AM was the first clue that these events were not solely dependent on NMDA activity. We have clarified the end of the first paragraph under the Results section to reflect this.

In order to more directly address the contribution of the NMDA receptor current to the observed Ca2+ transients we have added a new experiment as Figure 4F.

We incubated neurons with ryanodine for 15 minutes to block RyRs and then loaded them with Fluo-4 AM as before. These cells were imaged in Tyrode’s solution containing TTX but no extracellular Mg2+ to allow maximal NMDA currents. Under these conditions Ca2+ transients were observed, but when 1.25 mM Mg2+ was again added no further transients could be measured. These results illustrate that under physiological concentrations of Mg2+, Fluo-4 cannot detect the NMDA Ca2+ transient without further amplification from ER Ca2+ stores.

Furthermore, because Fluo-4 AM detects calcium events both in the presynapse and postsynapse, an explanation or experiment is required to distinguish these two sources. How did they define the calcium events as dendritic only?

In Figure 2D the addition of AP5 was able to abolish the vast majority of mSCTs as detected by Fluo-4 AM. This finding is evidence that these mSCTs are postsynaptic as the distribution of NMDA receptors is highly postsynaptic.

We used multiple probes or delivery methods to detect these mSCTs (postsynaptic dialysis, sparse tranfection of GCAMP5 probes etc) to verify the postsynaptic origin of the vast majority of the events. They are NMDA receptor block sensitive. Our earlier experiments in the same system provided little to no evidence for a role for presynaptic NMDA receptors during spontaneous neurotransmitter release (Atasoy et al., 2008 Supplementary Figure 2).

Figure 1B, is the impermeable Fluo-4 experiment conducted in Syb2-mO infected neurons? If so, it is worth checking if there is any spontaneous transients in the presynaptic terminal of cells that express Syb2-mO.

In our experiments to date we did not detect any pulsatile presynaptic Ca2+ release signals. In an earlier study, where we focused on presynaptic Ca2+ transients in presynaptic terminals (using a similar setting as in here), we found no evidence of discrete events but only of changes in baseline Ca2+ levels in response to neuromodulators such as Reelin (Refer to Bal et al., 2013 Neuron, Figure 3).

2) The authors showed mSCT frequency is nicely correlated with mEPSC frequency, when influenced by presynaptic players (Figure3). On the other hand intracellular calcium store blockers, Dantrolene and Ryanodine potentially only affecting the dendrite each reduce mSCT frequency (Figure 4). It is surprising and also hard to understand how calcium release from internal stores affects frequency rather than amplitude or kinetics of the calcium transients. It would be important to address if the presynapse is affected by Dantrolene and Ryanodine.

Calcium release from internal stores generates virtually all of the calcium signal visible with Fluo-4 imaging. The reduction in mSCT frequency with ER store blockers represents the fact that mSCTs can no longer be generated in their presence. In order to clarify this point, we have changed the wording in the Results section for Figure 4D and E (subsection “mSCT generation requires Ca2+ release from internal stores”). The remaining mSCTs after treatment with ryanodine are in fact of reduced amplitude suggesting that the remaining mSCTs are partially blocked by this treatment.

With 1.25 mM Mg 2+ in the extracellular solution the signal detected by Fluo-4 AM is exclusively produced by ER store calcium. To illustrate this point we conducted an experiment where cells were pre-treated with ryanodine to block calcium release from ER stores. Cells were then imaged with and without magnesium to show both the maximal current flow for the NMDA receptor (no magnesium) and the physiological condition (1.25 mM magnesium). With no magnesium in the bath and the NMDA receptor current uninhibited, we were able to measure calcium transients after the treatment with ryanodine. However when magnesium was again washed over the cells no further transients could be detected (Figure 4F). From these results we conclude that the diminutive NMDA current present in 1.25 mM magnesium is not detectable by our method.

3) Figure 5, the authors indicate that ryanodine and AP5 each only influence mEPSC amplitudes not the frequency. From the sample trace it appears that frequency is actually increased in both cases, a more representative trace is needed here.

As we indicated in our response to reviewer #2’s comments, the acute treatment of neurons with ryanodine did not alter mEPSC frequency. We have included this data to help illustrate that the primary effect of ryanodine in driving mEPSC scaling is the postsynaptic block of mSCT activity rather than driving a change in release frequency. This point has now been clarified in the Results section discussing Figure 5 (subsection “Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling”, second paragraph). Upon close inspection, the example trace shown in Figure 6A was not a good representative and has been replaced.

4) Why does ryanodine affect the calcium transient frequency, but affects only the mEPSC amplitude? This seems contradictory. I would expect an effect on the mEPSC frequency as well.

From our results, we believe that ryanodine acts downstream of mEPSC activity but its impact on generated Ca2+ transients and local protein translation alters mEPSC amplitudes. In other words, mSCTs largely report CICR driven Ca2+ signals that are triggered but not tightly coupled to the mEPSCs (which mainly report AMPA receptor driven currents). This issue is now better stated in the text where we discuss the impact of acute ryanodine treatment on mEPSC frequency (subsection “Blocking mSCTs induces homeostatic eEF2 kinase-dependent synaptic scaling”, second paragraph).

https://doi.org/10.7554/eLife.09262.013

Article and author information

Author details

  1. Austin L Reese

    Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    ALR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Ege T Kavalali

    1. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    ETK, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    ege.kavalali@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (NIH) (MH066198)

  • Ege T Kavalali

National Institute of Neurological Disorders and Stroke (NINDS) (T32 NS069562)

  • Austin L Reese

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank members of the Kavalali and Monteggia laboratories, in particular Dr Devon Crawford and Erinn Gideons for insightful discussions and comments on the manuscript. We would also like to thank Tom Reese for his assistance in streamlining the data analysis. This work was supported by NIH grants MH066198 (ETK) and the Cellular Biophysics of the Neuron Training Program T32 NS069562 (ALR).

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the UT Southwestern Medical Center (APN# 0866-06-05-1).

Reviewing Editor

  1. Indira M Raman, Northwestern University, United States

Publication history

  1. Received: June 6, 2015
  2. Accepted: July 24, 2015
  3. Accepted Manuscript published: July 24, 2015 (version 1)
  4. Version of Record published: August 13, 2015 (version 2)

Copyright

© 2015, Reese and Kavalali

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,878
    Page views
  • 513
    Downloads
  • 20
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Neuroscience
    Jennifer S Sun et al.
    Research Article
    1. Human Biology and Medicine
    2. Neuroscience
    Garron T Dodd et al.
    Research Article