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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2009 April 1.
Published in final edited form as:
PMCID: PMC2578837
NIHMSID: NIHMS71896

Spontaneous and evoked glutamate release activate two populations of NMDA receptors with limited overlap

Abstract

In a synapse, spontaneous and action potential-driven neurotransmitter release are assumed to activate the same set of postsynaptic receptors. Here, we tested this assumption using MK-801, a well-characterized use-dependent blocker of NMDA receptors. NMDA receptor-mediated spontaneous miniature excitatory postsynaptic currents (NMDA-mEPSCs) were substantially decreased by MK-801 within 2-minutes in a use-dependent manner. In contrast, MK-801 application at rest for 10-minutes did not significantly impair the subsequent NMDA receptor-mediated evoked EPSCs (NMDA-eEPSCs). Brief stimulation in the presence of MK-801 significantly depressed evoked NMDA-eEPSCs but only mildly affected the spontaneous NMDA-mEPSCs detected on the same cell. Optical imaging of synaptic vesicle fusion showed that spontaneous and evoked release could occur at the same synapse albeit without correlation between their kinetics. In addition, modeling glutamate diffusion and NMDA receptor activation revealed that postsynaptic densities larger than ~0.2 μm2 can accommodate two populations of NMDA receptors with largely non-overlapping responsiveness. Collectively, these results support the premise that spontaneous and evoked neurotransmission activate distinct sets of NMDA receptors and signal independently to the postsynaptic side.

Keywords: Synaptic vesicle release, synaptic transmission, synaptic communication, Synapse, NMDA receptor, Synaptic plasticity, neurotransmission

Introduction

Spontaneous synaptic vesicle fusion is a salient feature of all synapses (Fatt and Katz, 1952; Del Castillo and Katz, 1954) including those in synaptic networks in vivo (Pare et al., 1997; Pare et al., 1998; Chadderton et al., 2004). These random release events typically activate receptors within a single postsynaptic site and give rise to miniature postsynaptic currents, and therefore they have been extremely instrumental in analysis of unitary properties of neurotransmission. Under most circumstances the two forms of release occur concurrently without significant difference in their unitary properties (Isaacson and Walmsley, 1995; Wall and Usowicz, 1998). Spontaneous release typically occurs with a rate of 1 to 2 vesicles per minute per release site (Geppert et al., 1994; Murthy and Stevens, 1999; Sara et al., 2005) whereas evoked release at individual synapses can occur at an extremely high rate (>100 vesicles per second) (Saviane and Silver, 2006). However, it is yet unclear if spontaneous neurotransmitter release serves a well-defined purpose (Otsu and Murphy, 2003; Zucker, 2005). Despite lack of direct evidence for a physiological significance, several studies have shown that the blockade of spontaneous activation of neurotransmitter receptors causes independent or additional downstream effects compared to the blockade of evoked neurotransmission alone (McKinney et al., 1999; Sutton et al., 2004; Sutton et al., 2006).

How do postsynaptic neurons distinguish evoked and spontaneous neurotransmission and differentially activate postsynaptic signaling? To address this question, we examined the possibility that spontaneous and evoked neurotransmission activate non-overlapping postsynaptic NMDA receptor populations. For this purpose, our choice to examine NMDA receptor mediated synaptic responses was motivated by two reasons. First, recent findings indicate that NMDA receptors signal at rest (Sutton et al., 2006), in addition to their critical role in action potential triggered synaptic signaling. Second, working with NMDA responses enabled us to take advantage of MK-801, a well-characterized use-dependent blocker of NMDA receptors (Huettner and Bean, 1988; Hessler et al., 1993; Rosenmund et al., 1993). In this way, we could show that MK-801 dependent blockade of spontaneous NMDA-mEPSCs does not cause significant block of subsequent evoked NMDA-eEPSCs and vice versa. Optical imaging of spontaneous and evoked fusion kinetics at individual synapses supported the premise that both forms of release occur from the same synapse. Therefore we propose that spontaneous and evoked fusion events activate two populations of NMDA receptors with limited overlap, which may enable them to trigger independent postsynaptic signaling events.

Materials and Methods

Cell culture

High density dissociated hippocampal cultures were prepared from 1 day old Sprague-Dawley rat pups or wild type and synaptotagmin-1 deficient mouse pups using previously described methods (Kavalali et al., 1999). Autaptic hippocampal cultures were prepared as previously described (Mennerick et al., 1995).

Slice preparation

Transverse hippocampal slices (400 μM) were prepared from 12- to 21- day old Sprague–Dawley rats and incubated in oxygenated solution containing in mM: 124 NaCl, 5 KCl, 12 NaH2PO4, 26 NaHCO3, 10 D-Glucose, 2 CaCl2 and 1 MgCl2 at room temperature (24-27°C). For electrophysiological experiments, after making a cut between CA1 and CA3 regions, individual slices were transferred to the recording chamber that was mounted on the stage of an upright microscope (Nikon E600FN, Tokyo, Japan). During experiments, slices were submerged and extracellular solution was continuously exchanged with a flow rate of 1-3ml/min. All experiments were performed at room temperature.

Electrophysiology

In dissociated cultures, cells with pyramidal morphology were whole cell voltage-clamped to -70 mV in a modified Tyrode’s solution containing (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 10 glucose, 10 HEPES, and 2 CaCl2 (pH 7.4, 310 mOsm). To record and isolate NMDA receptor-mediated miniature or evoked EPSCs, MgCl2 concentration is reduced to 0 or 0.1 mM and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10μM, Sigma-Aldrich Co., St. Louis, MO, USA), picrotoxin (PTX; 50 μM; Sigma), strychnine (1 μM; Sigma), glycine (15 μM; Sigma) were added to bath solution. Electrode solution contained (in mM): 115 Cs-MeSO3, 10 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 20 TEA.Cl, 4 Mg-ATP, 0.3 Na2GTP, 10 QX-314 (Sigma, St Louis, MO, pH 7.35, 300 mOsm). Data was acquired using an Axopatch 200B amplifier and Clampex 8.0 software (Axon Instruments, Union City, CA). Recordings were filtered at 2 kHz and sampled at 5 kHz. To elicit evoked responses, electrical stimulation was delivered through parallel platinum electrodes in modified Tyrode solution (1 ms duration, 20-30 mA amplitude). Baseline for the analysis of NMDA-mEPSCs was automatically determined as the average current level of silent episodes during a recording. The events were selected at a minimum threshold of 4 pA and the area under current deflection was calculated to quantify charge transfer. Although, in most cases we quantified spontaneous NMDA-mEPSCs as charge transfer, we typically evaluated the reduction in evoked NMDA-eEPSCs in terms of their amplitudes. When we quantified evoked NMDA-eEPSCs as charge transfer (e.g. in Figures 2, ,44 and and8),8), this analysis did not result in a significant difference in the estimation of relative block of NMDA-eEPSCs compared to NMDA-mEPSCs or vice versa.

Figure 2
MK-801 treatment at rest minimally affects subsequent evoked NMDA-eEPSCs in hippocampal cultures
Figure 4
Differential MK-801 block and recovery of NMDA-eEPSCs and NMDA-mEPSCs
Figure 8
Modeling glutamate diffusion reveals geometric constraints on independent NMDA receptor signaling in individual postsynaptic densities

Whole-cell recordings from autaptic neurons were performed using an Axopatch 1D amplifier, a Digidata 1200 acquisition board, and pClamp software, version 9 (Axon Instruments, Foster City, CA). Electrodes had resistances of 3-5 MΩ, and access resistance was compensated 80-100%. In all instances, cells were excluded from analysis if a leak current > 300 pA was observed. For recording, the culture medium was exchanged for a saline solution containing (in mM): 138 NaCl, 4 KCl, 3CaCl2, 0 MgCl2, 10 glycine, 10 glucose, 10 HEPES, 0.05 bicuculline, and 0.001 NBQX (pH 7.25). The whole-cell pipette solution contained (in mM): 140 K-gluconate, 0.5 CaCl2, 5 EGTA, and 10 HEPES (pH 7.25). For synaptic recordings, cells were stimulated with 1.5 ms pulses to 0 mV from -70 mV to evoke transmitter release (Mennerick et al., 1995). Solutions were exchanged via a local multibarrel perfusion pipette with a common perfusion port placed within 0.5 mm of the cell under study. Liquid junction potential measurements exhibit exchange times between barrels in the order of ~50 ms. NMDA-mEPSCs were recorded in the presence of 500 nM TTX and analyzed with MiniAnalysis version 6.0 (Synaptosoft, Decatur, GA).

In hippocampal slices we recorded from the neurons in the pyramidal cell layer of area CA1 in the whole cell voltage-clamp configuration. Pipettes were filled with the internal pipette solution that contained (in mM): 110 K-gluconate, 20 KCl, 10 NaCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP, 0.3 GTP, 10 QX-314 and buffered to pH 7.3 with CsOH (290 mOsm). eEPSCs were evoked by stimulation (200 μs duration, 10-80μA amplitude) of Schaffer Collateral afferents using concentric bipolar tungsten electrodes through a stimulus isolation unit. All statistical comparisons were performed with two-tailed unpaired t-test (except in cases where measurements were paired e.g. Figures 2D, ,3B);3B); values are given as mean ± SEM.

Figure 3
Cross talk between NMDA-eEPSCs and NMDA-mEPSCs in hippocampal slices

Fluorescence detection of spontaneous and evoked release kinetics

High density dissociated cultures were infected with synaptophysin-pHluorin lentivirus at 8 days in vitro and analyzed at 13-14 days in vitro (synaptophysin-pHluorin construct contained 2 pHluorins and was a generous gift of Drs. Y. Zhu, and C.F. Stevens). Following 1 minute recording of baseline fluorescence, we perfused cultures with 10 nM freshly prepared folimycin. At high concentrations (~100 nM) folimycin increased background alkalinization independent of fusion, therefore in these experiments we used 10 nM folimycin, which uncovered spontaneous Ca2+-dependent alkalinization as expected from spontaneous fusion. Cultures were then allowed to rest for 10 minutes in the presence of 10 μM CNQX, 50 μM AP5 and 50 μM PTX to estimate spontaneous fusion rate. In these cultures, presence of CNQX impairs spontaneous action potential firing thus eliminates the need for TTX application (data not shown). Accordingly, the extent of spontaneous alkalinization in the presence of TTX (2 mM Ca2+) reached 21.2% of the total pool (n = 2 experiments, 70 boutons) compared to 22.4 % of the total pool reached in 2 mM Ca2+ in CNQX (see Figure 6). Afterwards, cultures were stimulated with parallel field electrodes (25 mA-1ms) at 1 Hz frequency for 10 minutes. At the end of the 10-minute period, cultures were exposed to 8 mM Ca2+ and stimulated maximally at 30Hz for 600 pulses to identify functional synaptic vesicle clusters. Images were acquired with a cooled CCD camera (CoolSnapHQ, Roper Scientific) during illumination (100 ms) at 480 ± 20 nm (505 dichroic long pass and 534 ± 25 bandpass) via an optical switch (Sutter Instruments) and analyzed using Metafluor Software (Universal Imaging).

Figure 6
Optical analysis of spontaneous and evoked release with synaptophysin-pHluorin at individual synapses

Modeling glutamate diffusion in the synaptic cleft and resulting NMDA receptor activation

Our glutamate diffusion model followed the approach previously used by Nielsen et al. (2004) and simulated isotropic diffusion of 4000 glutamate molecules (e.g. Xu-Friedman and Regehr, 2003) released from a point source. The glutamate molecules diffuse within a 20 nm gap of the cleft that has a geometric dimension of 600 nm × 600 nm × 20 nm. Once they are out of the cleft, they can rapidly diffuse in a large space. The geometry is described by a 3-dimensional matrix S = (Si,j,k), Si,j,k = 1 if the element is inside the domain of diffusion, Si,j,k = 0 if the element is outside of the domain i.e., in non-diffusible domain.

The standard thermo-diffusion equation is applicable for the glutamate concentration Ci,j,k = C(xi,yj,zk,t):

equation M1
(1)

where the diffusion constant Dglut takes the value 0.4 μm2/ms inside the cleft and 0.75 μm2/ms outside the cleft. Following the explicit finite-difference scheme, equation (1) can be transformed into the form:

equation M2
(2)

where

equation M3

and equation M4 is the concentration at next time node.

We take dx=0.01 μm and dt=0.02 μs. The space step and time step are chosen to ensure that the Courant–Friedrichs–Lewy condition (CFL condition) (Courant et al. 1927; LeVeque 2007) is satisfied and the explicit finite difference scheme will be numerically stable. Once the time course of glutamate distribution is obtained, we use a “3C2O” model with 2-coupled closed states for receptor kinetics (Popescu et al, 2004):

equation M5

We only consider the M-mode and L-mode kinetics for NMDA receptor. The population of each of the states are represented by their probability p1 (t), p2 (t), p3 (t), p4 (t), p5 (t), p6 (t), p7 (t). The open probability of popen (t) = p6 (t) + p7 (t). The populations pi (t) in M-mode satisfy a system of ordinary differential equations

equation M6
(3a)
equation M7
(3b)
equation M8
(3c)
equation M9
(3d)
equation M10
(3e)
equation M11
(3f)
equation M12
(3g)

The initial value is (p1 (t), p2 (t), p3 (t), p4 (t), p5 (t), p6 (t), p7 (t)) |t=0 = (1,0,0,0,0,0,0)

The glutamate concentration C(x, y, z, t) is taken at the location (x, y, z) where the receptor is located (averaged over 10 μs intervals). For L-mode kinetics, the equations are similar except for the reaction rate constants were chosen according to the following kinetic scheme:

equation M13

Results

NMDA-mEPSCs are rapidly blocked by MK-801

To test whether spontaneous and evoked synaptic vesicle fusion events activate the same set of postsynaptic receptors, we first pharmacologically isolated NMDA-receptor dependent synaptic responses by blocking AMPA and GABA receptors in the absence of extracellular Mg2+ and the presence of the NMDA receptor co-agonist glycine. We could clearly detect evoked and spontaneous NMDA-receptor mediated miniature EPSCs (NMDA-mEPSC) as judged by their sensitivity to AP-5, a selective blocker of NMDA receptors (Fig. 1A). For these experiments, we used dissociated hippocampal cultures since the kinetics of spontaneous synaptic vesicle fusion and recycling are well characterized in this system (Geppert et al., 1994; Ryan et al., 1997; Murthy and Stevens, 1999; Prange and Murphy, 1999; Sara et al., 2005; Virmani et al., 2005).

Figure 1
MK-801 block of NMDA-mEPSCs in hippocampal cultures

Application of 10 μM MK-801 rapidly blocked spontaneous NMDA-mEPSCs (Fig. 1B). In 12 days old hippocampal cultures, MK-801 induced block of NMDA-mEPSCs proceeded with a time constant of 24 seconds and reached a plateau within 60 seconds (Fig. 1C and 1E). In these measurements, we quantified charge transfer, a cumulative measure of NMDA receptor activity, as NMDA-mEPSCs are typically noisier than AMPA-mEPSCs (Fig. 1E inset). The kinetics of MK-801 block of spontaneous NMDA-mEPSCs is within a range expected from the previous estimates of the rate of spontaneous vesicle fusion in individual hippocampal synapses in culture (Geppert et al., 1994; Murthy and Stevens, 1999; Sara et al., 2005). The rate of block was further increased in older cultures (20 days in vitro) suggesting that frequency of spontaneous fusion per synapse increases during synapse maturation (Fig. 1D and 1E). At this developmental stage the MK-801 induced block reached completion within 30 seconds. Subsequent application of AP-5, a specific non-use-dependent blocker of NMDA receptors did not reduce the extent of baseline activity further (quantified as charge transfer) (Fig. 1F) verifying the completeness of the MK-801 block. In addition, to further test the use-dependent nature of the MK-801 block of NMDA-mEPSCs, we increased the extracellular Ca2+ from 2 mM to 8 mM, which causes up to 3-fold increase in the rate of spontaneous fusion (Fig. 1G, H and ref (Sara et al., 2005)). In 12 days old cultures, this increase in extracellular Ca2+ substantially increased NMDA-mEPSC activity as well as AMPA-mEPSCs and resulted in a 2-fold increase in the rate of MK-801 block of NMDA-mEPSCs (Fig. 1I).

In response to glutamate application NMDA receptors manifest relatively long latencies for opening. In addition, their openings typically outlast the duration of the glutamate pulse. Therefore, MK-801 application is expected to alter the kinetics of NMDA-mEPSCs as they occur (Jahr and Stevens, 1990; Lester et al., 1990; Hessler et al., 1993). In agreement with this expectation we detected a significant decrease in the decay times of clearly identifiable individual NMDA-mEPSCs after MK-801 application (Fig. 1J-1M). We also observed a small decrease in the rise times of NMDA-mEPSCs as their peaks became sharper in the presence of MK-801. This finding may suggest that individual NMDA receptors open asynchronously in response to glutamate release (Jahr, 1992)(Fig. 1J and 1M).

To verify that NMDA-mEPSCs were indeed triggered by vesicular glutamate release, we used folimycin, a specific blocker of vacuolar ATPase, which provides the proton gradient required for refilling synaptic vesicles with neurotransmitter. Application of 67 nM folimycin for 15 minutes decreased the activity of NMDA-mEPSCs as well as AMPA-mEPSCs to a similar extent (supplementary Fig. 1A, B), strongly indicating that both types of spontaneous activity were generated by similar vesicle fusion events. This finding makes it unlikely that non-vesicular glutamate release (for instance of astrocytic origin (Fellin et al., 2004)) could give rise to the NMDA-mEPSCs. This conclusion is also supported by several earlier studies that examined the properties of NMDA-mEPSCs in comparison to AMPA-mEPSCs (Umemiya et al., 1999; Groc et al., 2002; Dalby and Mody, 2003). We also evaluated the contribution of silent synapses (that solely contain NMDA receptors) to spontaneous neurotransmission in 20 DIV hippocampal cultures. For this purpose, we compared the frequency of AMPA-mEPCSs recorded at −70 mV to mixed AMPA/NMDA-mEPSCs recorded after relief of Mg2+ block at +40 mV on a given neuron (supplementary Fig. 1C-E). Under these conditions, we did not detect a significant difference between the frequency of AMPA/NMDA-mEPSCs measured at +40 mV (4.2 ± 0.7 Hz) and AMPA-mEPSCs measured at −70 mV (3.8 ± 0.5 Hz) (p>0.05, n=13 cells). This finding suggests that NMDA-mEPSCs that contribute to the activity at +40 mV are not significantly in excess of the AMPA-mEPSCs detected in isolation at −70 mV, indicating minimal contribution of silent synapses to spontaneous mEPSC activity in these cultures. In addition, we also tested if the rapid MK-801 block of NMDA-mEPSCs we observed above (Fig. 1) may in part be due to block of presynaptic NMDA receptors. To evaluate this possibility we assessed the impact of rapid extracellular MK-801 application on AMPA-mEPSCs in the absence of Mg2+ and found that the spontaneous fusion rate is only minimally sensitive to presynaptic NMDA receptor activity (see supplementary Fig. 2 for a detailed discussion).

MK-801 treatment at rest minimally affects subsequent evoked NMDA-eEPSCs

To quantify the cross talk between NMDA receptors that are activated in response to evoked versus spontaneous neurotransmitter release, we triggered release from a large fraction of synapses onto a neuron by invoking a single action potential driven NMDA-eEPSC using field stimulation (Fig. 2A and 2B). After recording the NMDA-eEPSC, we exchanged the medium with a solution containing 10 μM MK-801/1 μM TTX for 10 minutes to block spontaneous action potential firing and eliminate NMDA-mEPSCs. At the end of the 10-minute period, we rapidly washed out MK-801 and TTX and measured the size of the evoked NMDA-eEPSC on the same cell. As we have shown above, 10-minute perfusion of MK-801 is more than sufficient to block nearly all the NMDA-mEPSC activity on a given neuron (Fig. 2C). In contrast, the amplitudes of NMDA-eEPSCs recorded before and after the incubation period were not affected by the MK-801 treatment in between (before MK-801: 1072 ± 157 pA, after MK-801: 879 ± 114 pA, p>0.2, n=5) suggesting that application of MK-801 at rest did not block the NMDA receptors activated during stimulation (Fig. 2D). We also repeated the same experiment using weaker stimulation to decrease peak NMDA-eEPSC by 50% and activate only a fraction of the synapses. Under this condition we still observed a modest reduction in NMDA-eEPSC amplitude after 10-minute incubation with MK-801 (before MK-801: 489 ± 67 pA, after MK-801: 411 ± 29 pA, p>0.05, n=4). Under both strong and weak stimulation conditions, continued stimulation in the presence of MK-801 resulted in rapid use-dependent block of evoked NMDA-eEPSCs. The lack of cross talk between the block of evoked NMDA-eEPSCs and spontaneous NMDA-mEPSCs was not due to unblocking of NMDA receptors during wash out of TTX and MK-801 (~1 min) because use-dependent relief from MK-801 proceeds with a slow time course (Huettner and Bean, 1988). This was also evident in additional experiments a two-minute wash out of MK-801 did not induce significant unblocking of the NMDA-eEPSCs (Fig. 2A-2B). Here, it is important to note that a 10-minute-long whole cell recording results in some run down of the NMDA-eEPSC independent of MK-801 treatment. Therefore, the extent of reduction we detected in NMDA-eEPSCs was well within the variability of NMDA-eEPSC amplitudes detected during a stable recording (black circle in Fig. 2E). This finding is consistent with the original experiments, which documented the strict use dependence of MK-801 block and proposed the use of this compound to estimate the probability of neurotransmitter release (Hessler et al., 1993; Rosenmund et al., 1993). A small reduction in NMDA-eEPSCs after extended incubation with MK-801 is also in agreement with a recent study, which reported a 28% decrease in NMDA-eEPSCs after 15 minutes of MK-801 application (Scimemi et al., 2004).

Earlier experiments have also shown that evoked NMDA receptor currents show slow spontaneous recovery (~20 min) after MK-801 block (Tovar and Westbrook, 2002) implicating slow mobility of NMDA receptors on dendrites. Indeed, when we incubated hippocampal cultures with MK-801/TTX containing solution for 20 and 40 minutes and compared the sizes of NMDA-eEPSCs to NMDA-eEPSCs from vehicle treated control cultures, we detected a gradual reduction in the size of NMDA-eEPSCs reaching 70% by 40 minutes (Fig. 2E). Furthermore, we also detected a faster rate of mixing between the two populations of receptors at 32°C presumably due to increased mobility of NMDA receptors. At this temperature, the decrease in NMDA-eEPSCs reached completion within 40 minutes after incubation with MK-801 and TTX (supplementary Fig. 3). These results suggest that the NMDA receptors activated by spontaneous release events may mix with other NMDA receptors over long periods consistent with the slow mobility and mixing of NMDA receptors on a dendrite. In addition, our experiments do not fully exclude other possibilities such as insertion of new NMDA receptors or a very slow rate of spontaneous release at the same location with evoked release.

Cross talk between NMDA-eEPSCs and NMDA-mEPSCs in hippocampal slices

We next examined whether these observations obtained in dissociated hippocampal cultures were valid in situ using an acute hippocampal slice preparation. One advantage of the hippocampal slice preparation is that in the absence of stimulation most of the spontaneous events can be attributed to true spontaneous release even in the absence of TTX. This is because once a cut is introduced between the CA3-CA1 regions of a slice the propensity of spontaneous action potential driven activity diminishes. First, we replicated the original observations in hippocampal slices (Hessler et al., 1993). Namely, a 10-minute application of 50μM MK-801 after an initial evoked NMDA-eEPSC did not occlude the subsequent evoked NMDA-eEPSC in the continued presence of MK-801 (Fig. 3A and 3B). However, the second evoked NMDA-eEPSC declined rapidly (Fig. 3A) as expected from the block of open NMDA channels by MK-801 during the NMDA-eEPSC. In contrast, spontaneous NMDA-mEPSCs detected in the interim period showed swift biphasic block in agreement with the previous results from the cultures (Fig. 3C and 3D). The fast component of the decay had a time constant of 19 seconds whereas the slow component reached completion by the end of a 10-minute period of MK-801 exposure (down to 4% of the initial activity). This finding suggests that in slices, the spontaneous fusion rate of approximately 50% of the synapses is rather slow (τ=245 s) compared to dissociated cultures. After a ten-minutes long washout of MK-801, NMDA-mEPSCs showed only 9 ± 3% (n = 4) recovery consistent with their successful block during MK-801 application. These findings indicate that in acute hippocampal slices, as in cultures, the NMDA receptor population activated by spontaneous fusion events does not overlap with the one that gives rise to evoked NMDA-eEPSCs.

MK-801 block of NMDA-eEPSCs has minimal effect on subsequent NMDA-mEPSCs detected on the same cell

If the fusion pore kinetics or glutamate release profile of the spontaneous and evoked fusion events in a given synapse differed dramatically, then one can surmise a scenario where evoked fusion events may reach a higher percentage of receptors whereas spontaneous fusion events may activate only a very small number of receptors (Cull-Candy and Leszkiewicz, 2004). This setting could give rise to an apparent lack of overlap between the receptor pools activated by the two forms of release, as block of NMDA-mEPSCs would have a negligible effect on evoked NMDA-eEPSCs. To evaluate this possibility, we tested whether use-dependent block of evoked NMDA-eEPSCs impair subsequent NMDA-mEPSCs using field stimulation in the hippocampal culture system to activate most synapses on a neuron (Fig. 4A). After recording NMDA-mEPSCs on a given cell, we rapidly perfused MK-801 after washing out TTX and stimulated cultures with 30 action potentials applied at 3 Hz (10 seconds). This moderate stimulation was used to minimize potential glutamate spill over. We then rapidly removed MK-801 and measured the NMDA-eEPSC after 1 minute (Fig. 4B). This delay was necessary as this stimulation paradigm induces some synaptic depression independent of the MK-801 block, therefore the actual extent of block was measured once synapses recover from this weak depression. Under these conditions, we detected a 61.6 ± 6 % reduction in NMDA-eEPSCs and only a 23.4 ± 16.5 % reduction in NMDA-mEPSCs (Fig. 4C). Application of MK-801 for 10 seconds without stimulation could induce a 15.5 ± 6.6 % reduction in NMDA-mEPSCs by itself indicating most of the reduction seen in spontaneous NMDA-mEPSCs cannot be attributed to block due to evoked release despite some potential for spill-over during the 3 Hz stimulation (Fig. 4C). In addition, there is some variability in NMDA-mEPSC activity measured over 10 second intervals (-2.8 ± 10.9 % (n = 4)), which should be taken into account in comparison of these results (Fig. 4C).

Differential use-dependent recovery of NMDA-eEPSCs and NMDA-mEPSCs from MK-801 block

After removal of MK-801, the recovery of NMDA receptors from MK-801 block is also use-dependent, as it requires the presence of glutamate and proceeds with a time constant of 90 minutes at −70 mV (Huettner and Bean, 1988). In the next set of experiments, we tested whether NMDA-mEPSCs recover from MK-801 block and if this recovery is coupled to recovery of NMDA-eEPSCs. In these experiments, we first measured the extent of NMDA-mEPSC activity on a given neuron in the presence of TTX and after TTX wash out measured evoked NMDA-eEPCSs at 0.2 Hz stimulation for 10 pulses (Fig. 4D and 4E). At this point, we perfused MK-801 during stimulation for another 5 minutes until evoked responses were diminished. To ensure full block of NMDA-mEPSCs, we re-perfused TTX and MK-801 and quantified the depression of NMDA-mEPSCs for 2 minutes. This procedure guaranteed substantial block of both evoked NMDA-eEPCSs and spontaneous NMDA-mEPSCs. After removal of MK-801, we monitored the recovery of spontaneous NMDA-mEPSCs for 10 minutes (Fig. 4E). After 10 minutes, NMDA-mEPSCs showed 27.8 ± 6.7 % recovery whereas at this time point, evoked NMDA-eEPSCs (measured after TTX wash out) have recovered only 13.1 ± 3.9% of their initial amplitudes (n = 5, p < 0.05) (Fig. 4F). This finding indicates that activation of NMDA receptors at rest by spontaneous glutamate release is not sufficient to remove MK-801 block of NMDA receptors activated in response to evoked release.

To further examine the extent of cross talk between NMDA receptors activated by evoked and spontaneous release, we recorded NMDA receptor-mediated events in solitary-neuron cultures in which neurons form recurrent autaptic connections onto themselves. The main advantage of this preparation is that a single action potential generated via intracellular stimulation applied to the cell body of a neuron can access all synapses thus minimize the possibility of artifacts that may originate from selective activation of synapses by field stimulation (Mennerick et al., 1995). First, we assessed the effect of blocking NMDA-mEPSCs with MK-801 on subsequent evoked NMDA-eEPSCs (Fig. 5A). eEPSCs and mEPSCs were recorded prior to a 20 min incubation in 5 μM MK-801 and 500 nM TTX (Fig. 5B). At the end of the 20-minute period, MK-801 was washed out and NMDA-eEPSCs and NMDA-mEPSCs were again recorded (Fig. 5A, B). 20 minutes in MK-801 was sufficient to eliminate almost all spontaneous NMDA-mEPSCs (Fig. 5C), but similar to high-density hippocampal cultures, the decrease in evoked NMDA-eEPSC peak amplitudes was not a statistically significant (p > 0.37) (Fig. 5D). The percent reduction in NMDA-mEPSCs was significantly higher than the decrease in NMDA-eEPSCs (p< 0.01) (Fig. 5E). Second, we performed the counter experiment: we first blocked evoked NMDA-eEPSCs with MK-801 and determined if NMDA-mEPSCs were also affected (Fig. 5F). For this purpose, we initially recorded mEPSCs for 1 min, just prior to a 30-pulse stimulus train at 3 Hz in the presence of MK-801 (Fig. 5G). After the train, MK-801 was washed out for 1 min, and then another evoked NMDA-eEPSC was recorded, followed by the recording of NMDA-mEPSCs for another minute (Fig. 5G). In this setting, we detected a 50.6 ± 7.2% decrease in NMDA-eEPSCs (Fig. 5H), while the decrease in NMDA-mEPSCs was only 24.2 ± 4.4% (Fig. 5I). Although MK-801 application decreased the amplitudes of NMDA-eEPSCs as well as the NMDA-mEPSCs, the percentage of reduction in NMDA-eEPSCs was higher than the decrease in NMDA-mEPSCs (p < 0.05) (Fig. 5J). This finding is consistent with the experiments performed using field stimulation of high-density cultures (see Fig. 4A-C).

Figure 5
Cross talk between NMDA-eEPSCs and NMDA-mEPSCs in autaptic cultures

Optical analysis of spontaneous and evoked neurotransmission at individual synapses reveals no correlation between the kinetics of the two forms of release

If synapses capable of efficient evoked neurotransmitter release were poor in spontaneous release and vice versa, then one would expect to see a lack of cross talk between the sets of NMDA receptors activated by the two forms of release. In the next set of experiments, we addressed this question by using an optical imaging approach to compare the propensity of evoked and spontaneous release at individual synapses. For this purpose, we infected hippocampal cultures with lentivirus containing DNA for the synaptic vesicle protein synaptophysin tagged with superecliptic pHluorin (synaptophysin-pHluorin), a GFP-based pH sensor that is normally quenched at pH 5.5 within the vesicle lumen but fluoresces once vesicles fuse and the fluorophore is exposed to extracellular pH (7.4) (Miesenbock et al., 1998). This construct is better localized to synaptic vesicles and manifests minimal surface fluorescence compared to the classical synaptophluorin, which is a fusion construct of synaptobrevin (Zhu et al., Society for Neuroscience Abstract 2004; (Granseth et al., 2006)) (Fig. 6A). For these imaging experiments we selected isolated 1 μm2 fluorescent puncta, which typically correspond to individual synapses (Liu et al., 1999). To correlate the rate of spontaneous release with evoked release we incubated cultures with a low concentration of folimycin (10 nM) a high-affinity blocker of vacuolar ATPase required for re-acidification of vesicles upon endocytosis (Drose and Altendorf, 1997). Folimycin traps vesicles in an alkaline state after endocytosis and provides a cumulative measure of exocytosis (Ryan et al., 1997; Sankaranarayanan and Ryan, 2001). At rest, using 10 nM folimycin we detected a slow Ca2+-dependent increase in fluorescence due to spontaneous fusion activity (Fig. 6A and 6B). At 0 mM Ca2+ the increase in fluorescence was nominal, in contrast, increasing Ca2+ concentration to 8 mM resulted in clearly perceptible rise in fluorescence (Fig. 6B). Thus this spontaneous fluorescence increase was sensitive to alterations in extracellular Ca2+ concentration similar to spontaneous neurotransmitter release and FM dye release detected in the same system (Fig. 1G-H) (Sara et al., 2005). We also tested this premise by quantifying the rate of fluorescence increase 3 minutes after application of saturating stimulation (30 Hz, 2 minutes) to mobilize most of the recycling vesicles in the presence of folimycin in 2 mM Ca2+. The rate of fluorescence increase due to spontaneous alkalinization under this condition was not significantly different from the alkalinization rate at 0 mM Ca2+ (p>0.5) (n=3 experiments, 366 boutons)(Fig. 6B-inset). In addition, the sites of spontaneous fluorescence increase coincided with the locations sensitive to intense action potential stimulation (600 pulses at 30 Hz) indicating that these sites corresponded to presynaptic boutons (Fig. 6B).

The kinetics of vesicle mobilization at 1 Hz is dependent on the probability of neurotransmitter release in individual synapses (Waters and Smith, 2002). This is mainly because at 1 Hz stimulation there is very little synaptic depression and thus each stimulation gives rise to independent trials. Therefore, in the next set of experiments we monitored the rate of spontaneous fluorescence rise in tandem with the rise in fluorescence in response to 1 Hz stimulation (Fig. 6C and 6D). This paradigm was followed by 30 Hz stimulation in the presence of 8 mM Ca2+ in order to clearly identify synaptic boutons. Under these conditions, we analyzed a total of 440 boutons (9 experiments) by measuring the rate of fluorescence rise from the linear portions of individual traces for spontaneous and evoked recording segments (Fig. 6C and 6D). Restricting our measurements to the linear portions of the traces was necessary because a significant number of the traces reached a plateau presumably due to reuse of vesicles in an alkaline-trapped state (Fig. 6C).

Overall, the comparison of release rates did not reveal a significant positive or negative correlation between the propensities for evoked and spontaneous fusion of individual synapses (R=0.001) (Fig. 6E). 14.7% (65 synapses) of synapses exhibited either no evoked release or they showed evoked release at a rate less than spontaneous release (Kimura et al., 1997). In 79% of the synaptic boutons evoked release kinetics was equal or faster than that of spontaneous release. 6% (27 synapses) did not show any detectable spontaneous release but manifested evoked release. Furthermore, 3 boutons showed no release under both conditions but responded to 30 Hz stimulation dramatically. On average, evoked release rate per minute at 1 Hz was 11.9 times faster than the spontaneous release rate. The median of the fold increase in release rate during 1 Hz stimulation was 5.4. If we assume an evoked release probability of 0.2 for hippocampal synapses in culture (Murthy et al., 1997) then we would expect release of approximately 12 vesicles during 60 seconds of 1 Hz stimulation. From the ratio of evoked and spontaneous release rates, we can estimate the spontaneous release rate for a typical synapse as 1 to 2 vesicles per minute. This estimate is in line with earlier work (Geppert et al., 1994; Murthy and Stevens, 1999; Sara et al., 2005) as well as our electrophysiological measurements in this study (Fig. 1E). Taken together, this analysis suggests that spontaneous and evoked release occur within the same synapses (79% of the time) however, without significant correlation between their kinetics. In addition, spontaneous release events do not appear to be ectopic as they occur at discrete spots coincident with intense evoked release rather than being diffusely distributed along an axon.

Differential sensitivity of NMDA-eEPSCs and NMDA-mEPSCs to MK-801 block induced by brief NMDA application

To further explore the differential properties of NMDA receptors activated in response to evoked versus spontaneous release, we co-applied 100 μM NMDA and 30 μM MK-801 (in 15 μM glycine) to hippocampal cultures and tested the block of global NMDA responses, spontaneous NMDA-mEPSCs and evoked NMDA-eEPSCs (Fig. 7A). 5-second application of NMDA-MK-801 cocktail was sufficient to reduce the global NMDA response by 95±0.6% (n=5) (Fig. 7B-C). Under the same condition, spontaneous NMDA-mEPSC activity was reduced by 97±0.8% (n=6) (Fig. 7D). In contrast, evoked NMDA-eEPSCs were mildly affected by this treatment as they were only reduced by 45±8% (n=14) (49.6+/-5 % in terms of peak amplitudes, mean peak amplitude before MK801 = 1499.8 +/- 197 pA after MK801 = 755+/-136 pA.) (Fig. 7E). Subsequent application of AP-5 could completely block this evoked current, whereas it had nominal effect on the remaining baseline activity (data not shown). Furthermore, remaining NMDA-eEPSCs showed minimal recovery from MK-801 block during 5 pulses applied at 0.1 Hz (data not shown). Prolongation of the NMDA-MK-801 cocktail perfusion to 30 seconds caused substantial block of all forms of NMDA receptor activity (>91%, n=7). Similarly, increasing NMDA concentration to 1 mM resulted in a substantial block of all NMDA receptor mediated activity within 5 seconds (>90%, n= 5) (Fig. 7F-I). This final result suggests that NMDA concentration was the predominant limiting factor in the partial block of NMDA-eEPSCs consistent with the 10-30 μM affinity of NMDA receptors for NMDA (Patneau and Mayer, 1990). Taken together, these results indicate that briefly applied exogenous 100 μM NMDA has limited ability to activate receptors that mediate evoked NMDA-eEPSCs whereas NMDA receptors that sustain spontaneous neurotransmission can be readily activated by the same concentration of NMDA and thus strongly blocked by MK-801. These findings are consistent with the premise that NMDA receptors responding to evoked neurotransmission have a low probability for opening compared to the receptors responding to spontaneous neurotransmission. This difference in open probabilities may be due differential subunit composition of receptors responding to evoked and spontaneous release. For instance, NMDA receptors composed of NR1/NR2B subunits display higher affinity fro the coagonist glycine and slower deactivation (Priestley et al., 1995; Thomas et al., 2006). However in a separate set of experiments, we did not detect a significant difference between the compositions of NMDA receptors activated by the two forms of release as they were both equally blocked by the NR2B specific antagonist ifenprodil (3 μM) by 50% [(NMDA-eEPSCs before ifenprodil: 1412 ± 299 pA; after ifenprodil: 695 ± 167pA; n = 5: p = 0.019) (NMDA-mEPSCs before ifenprodil: 83.2 ± 22 pC/10s; after ifenprodil: 47.7 ± 13 pC/10s; n = 5; p = 0.003)]. Taken together these findings provide additional support to the notion that spontaneous and evoked glutamate release activates distinct populations of NMDA receptors.

Figure 7
Relatively higher susceptibility of spontaneous NMDA-mEPSCs to MK-801 block during exogenous NMDA application compared to evoked NMDA-eEPSCs

Quantitative assessment of geometric constraints on independent NMDA receptor signaling at individual postsynaptic densities

The results presented so far argue for the co-existence of evoked and spontaneous release within a 1 μm2 synaptophysin-pHluorin labeled spot that presumably correspond to individual presynaptic terminals. This finding is consistent with earlier work performed using uptake and release of FM dyes (Sara et al., 2005; Groemer and Klingauf, 2007; Prange and Murphy, 1999), and postsynaptic Ca2+ imaging (Murphy et al., 1994; Murthy et al., 2000). Given the area covered by a single postsynaptic density (PSD) can range between 0.07 μm2 (~260 nm × 260 nm) to 0.42 μm2 (~650 nm × 650 nm) (Harris and Sultan, 1995), we next estimated the minimum distance between two sets of NMDA receptors that respond to glutamate release in such a restricted area and have limited cross talk. To address this question, we modeled glutamate diffusion in the synaptic cleft and resulting NMDA receptor activation upon fusion of a single vesicle. Our glutamate diffusion model followed the approach previously used by Nielsen et al. (2004). In this model, we simulated isotropic diffusion of 4000 glutamate molecules released from a point source in a simplified synaptic cleft with a 20 nm distance between the presynaptic and postsynaptic regions that both cover a 600 nm × 600 nm area (0.36 μm2) (Fig. 8A, B). This large area enabled us to assess glutamate diffusion and NMDA receptor activation at several locations with respect to the initial point of release and helped us visualize key geometric requirements for limited cross talk between NMDA receptor populations. Within the synaptic cleft we assumed a diffusion coefficient of 0.4 μm2/ms in accordance with several earlier estimates (Rusakov and Kullmann, 1998; Xu-Friedman and Regehr, 2003; Nielsen et al., 2004), whereas outside the cleft we assumed a diffusion coefficient of 0.75 μm2/ms. The latter represents the diffusion coefficient for glutamate in free solution (Xu-Friedman and Regehr, 2003). Thus, once glutamate molecules leave the cleft, they can diffuse in a large region with a faster rate. In this model, we did not incorporate glutamate buffering by transporters, as our experiments showed that application of 50 μM TBOA, a potent wide spectrum blocker of glutamate transporters, did not lead to a cross talk between spontaneous NMDA-mEPSC activity and subsequent evoked NMDA-eEPSCs (10 minutes of incubation with TTX+MK-801+TBOA decreased NMDA-eEPSCs only to 97.1±11 % of their initial amplitude, n=5). However, TBOA treatment caused a 3.18±07-fold increase in NMDA-mEPSCs (n=5) suggesting that these transporters are active and may limit glutamate diffusion at rest.

To estimate the degree of NMDA receptor activity at various locations, we equally spaced 16 NMDA receptors within the 0.36 μm2 PSD area (R1 through R16 in Fig. 8A). Fig. 8C depicts the decrease in peak glutamate concentration following release of a single vesicle on top of receptor 6 (R6) (a receptor located near the center of the PSD) versus glutamate release on receptor 16 (R16) (a receptor at the periphery of the PSD). The distance between these receptors is 466 nm thus they are located at the opposite edges of a ~0.2 μm2 area. Irrespective of the release site (on R6 or R16) glutamate concentration decreases more than a 1000-fold once it reaches the other receptor (Fig. 8C).

To determine the peak open probabilities of NMDA receptors following glutamate release on R6 or R16, we used the NMDA receptor activation scheme proposed by Popescu and colleagues (2004). This scheme can account for several key features of synaptic NMDA receptor mediated currents including their non-saturation behavior seen in response to repetitive stimulation as well as their predominant modes of activity (High open probability H-mode, medium open probability M-mode and low open probability L-mode) (Popescu et al., 2004). Figure 8D depicts the decrease in open probabilities of receptors R16 through R1 following release on R16 (in M-mode or L-mode). This plot indicates that receptors at the opposite corner of the PSD at a distance of 466 nm and beyond (e.g. R13, R9 and R6 through R1) are far less likely to open (≥ 9-fold) in response to glutamate release on R16 (with the assumption that all receptors possess the same mode of activity, e.g. all in M-mode or L-mode). However, if these receptors respond to evoked release and typically manifest a low probability mode (such as the L-mode) as suggested by our experiments presented in Figure 7, and spontaneous release occurs in the vicinity of R16 (near the periphery of the PSD), then the receptors at the opposite corner of the PSD show a more than ~20-fold reduction in peak open probability compared to R16. This dramatic difference in peak open probabilities of the receptor populations can explain the finding that application of MK-801 at rest has very little effect on subsequent evoked responses (Figs. 2, ,3,3, 5A-E).

We next estimated the open probabilities of all receptors in response to glutamate release onto R6, which according to our model is the putative site of evoked release (Fig. 8E). In this case, if all receptors are in the same mode then R16 displays a 4.5-fold decrease in open probability compared to R6. However, the difference between the open probabilities of R16 and R6 decrease to ~1.9-fold if we follow the above assumption (and our results presented in figure 7) that R16 is M-mode and R6 is L-mode. This difference in peak open probabilities is compatible with the results of experiments where block of evoked NMDA responses results in a milder reduction subsequent spontaneous NMDA receptor activity (Fig. 4A-C, 5F-J).

Taken together, this model can recapitulate several key features (including the asymmetry in the extent of cross talk detected after MK-801 block of NMDA-mEPSCs versus NMDA-eEPSCs) with the assumption that within a 0.36 μm2 PSD a release event near the center (e.g. the vicinity of R6) represents evoked neurotransmission whereas a fusion event at the periphery of the PSD (e.g. near R16) corresponds to spontaneous release. Moreover, this model indicates that our experimental findings are in line with the commonly accepted parameters governing glutamate diffusion in synapses (Rusakov and Kullmann, 1998; Xu-Friedman and Regehr, 2003; Nielsen et al., 2004). According to this model medium to large (>0.2 μm2 area) synapses can easily accommodate independent signaling via spontaneous and evoked release with some geometric constraints. In a medium sized synapse, the two forms of release would need to be organized laterally (towards opposite corners of the synaptic cleft), whereas in a large synapse the independence of spontaneous and evoked neurotransmission can be achieved if the two forms of release are organized in a center-surround fashion. This model does not fully exclude the possibility that some synapses, especially small synapses (<0.2 μm2 area) which may be below the resolution of our optical experiments (shown in Fig. 6) can manifest solely spontaneous or evoked release and contribute to our electrophysiological observations. Furthermore, here we should note that deviations from the common assumptions we used in this model (such as a decrease in the number of glutamate molecules per vesicle, the peak glutamate concentration in the cleft upon fusion, incorporation glutamate transporters or other glutamate binding sites) could have a profound effect on the extent of cross talk between the two forms of release.

Asynchronous release detected in the absence of synaptotagmin 1 or in the presence of Sr2+ is resistant to MK-801 application at rest

Finally, we extended our observations by asking whether asynchronously released neurotransmitter quanta activate the same set of NMDA receptors as spontaneous release. To address this question we used hippocampal cultures from mice deficient in the synaptic vesicle protein synaptotagmin 1 (syt 1). Syt 1 deficient (syt 1 -/-) synapses release vesicles asynchronously, as syt 1 is the major sensor for the Ca2+-dependent synchronous release (Geppert et al., 1994; Fernandez-Chacon et al., 2001). In syt 1 -/- cultures, evoked NMDA-eEPSCs were significantly delayed in their activation and decay, consistent with earlier observations from evoked AMPA-eEPSCs in these cultures (Geppert et al., 1994) (Fig. 9A-B). Interestingly, in parallel with our observations in wild-type rat and mouse cultures, asynchronous NMDA-eEPSCs in syt 1 -/- cultures were unaffected by the 10 minute MK-801 treatment at rest (in the presence of TTX) (Fig. 9A and 9C). In contrast, in syt 1-/- cultures, the frequency of spontaneous NMDA-mEPSCs were two-fold higher than that of controls (Pang et al., 2006) and accordingly, their rate of MK-801 block was also significantly hastened compared to cultures from littermate controls (Fig. 9D and 9E) (wild type (wt), τ = 19 s, syt 1 -/-, τ = 8 s). These findings suggest that asynchronous release events detected in the absence of syt 1 activate that same set of NMDA receptors as synchronous events under control conditions. This result agrees with earlier studies showing that synchronous and asynchronous release originate from the same set of vesicles albeit with different release kinetics (Otsu et al., 2004; Sakaba, 2006). Furthermore, the increase in mEPSCs detected in syt 1-/- synapses is independent of the alteration in synchronicity of release seen in the absence of syt 1.

Figure 9
Asynchronous release detected in the absence of synaptotagmin 1 or in strontium is resistant to MK-801 application at rest

Asynchronous release can also be triggered in wild type neurons when Sr2+ is used instead of Ca2+ as the charge carrier to elicit neurotransmitter release (Dodge et al., 1969). Therefore, Sr2+ is commonly used in experimental settings where quantal events associated with a specific stimulated input onto a neuron is examined (Oliet et al., 1996). In rat hippocampal cultures, substitution of Ca2+ with Sr2+ resulted in a prominent asynchronous component of the AMPA-eEPSC (Fig. 9F). In contrast, the properties of NMDA-eEPSCs were not substantially altered presumably due to the high affinity of NMDA receptors to glutamate and the resulting slow kinetics of NMDAR-mediated currents (Fig. 9G). Perfusing cultures with MK-801 for 10 minutes (in 2 mM Ca2+) after eliciting a single Sr2+-mediated NMDA-eEPSC did not significantly alter the magnitude of the subsequent Sr2+-mediated NMDA-eEPSC (Fig. 9H, I). However, continued stimulation in the presence of Sr2+ and MK-801 for 3 minutes resulted in substantial inhibition of NMDA-eEPSCs (Fig. 9J) and washing out Sr2+ with Ca2+ did not cause significant recovery (Fig. 9J, K). Taken together, these findings suggest that, as in the case of syt 1-/- synapses, Sr2+ triggered asynchronous release does not activate the same NMDA receptors as spontaneous release and NMDA receptors activated by asynchronous and synchronous release overlap.

Discussion

In this study, we took advantage of MK-801, a high affinity use-dependent blocker of NMDA receptors, and found that there is limited cross talk between the NMDA receptors that are activated in response to spontaneous versus evoked glutamate release. This finding is based on four principle observations: First, electrophysiological experiments in high density and autaptic hippocampal cultures as well as hippocampal slices showed that use dependent block of spontaneous NMDA-mEPSCs and evoked NMDA-eEPSCs were largely independent. In these experiments MK-801 application caused a rapid block of NMDA-mEPSCs that was not mediated by the block of presynaptic NMDA receptors as their inhibition had minimal effect on the rate of spontaneous release. Second, once the NMDA receptors that are activated by both evoked and spontaneous release were blocked, NMDA-mEPSCs showed significant recovery at rest without concomitant recovery of NMDA-eEPSCs. Third, MK-801 block induced by brief exogenous NMDA application preferentially affected spontaneous NMDA-mEPSCs and partially spared evoked NMDA-eEPSCs suggesting that NMDA receptors responding to evoked neurotransmission have a low probability for opening compared to the receptors responding to spontaneous neurotransmission. Finally, modeling glutamate diffusion and NMDA receptor activation showed that synapses with PSDs larger than 0.2 μm2 could accommodate two populations of NMDA receptors than respond independently to two fusion events.

There are multiple scenarios that may account for these findings. First, spontaneous and evoked fusion events may originate from different synapses thus they may not activate the same set of receptors (Townsend et al., 2003). However, previous studies in hippocampal cultures have documented substantial co-localization of spontaneous and evoked synaptic vesicle recycling in individual synaptic boutons using uptake of fluorescent markers (Murthy and Stevens, 1999; Prange and Murphy, 1999; Murthy et al., 2000; Sara et al., 2005; Groemer and Klingauf, 2007). Furthermore, the same studies have shown that the sizes of the vesicle pools labeled with spontaneous versus evoked uptake of fluorescence probes in a given nerve terminal are strongly correlated (Murthy and Stevens, 1999; Prange and Murphy, 1999; Sara et al., 2005). Therefore, we consider complete segregation of spontaneous and evoked neurotransmitter release into different synapses as unlikely. Accordingly, optical analysis we performed in this study showed that at least 79% of the synapses are both capable of evoked and spontaneous release although the kinetics of the two forms of release were not correlated in a given synapse. However, a recent study in the retinal bipolar cell presynaptic terminals using total internal reflection fluorescence microscopy showed that spontaneous fusion events were largely excluded from synaptic ribbons which comprised the preferential site for evoked fusion (Zenisek, 2008). Therefore, we cannot exclude that some spontaneous and evoked fusion events may occur at different synapses. This possibility is hard to ascertain in our measurements due to two major caveats of our optical analysis. First, selection of fluorescence puncta in dissociated hippocampal cultures typically favors large synapses over small ones. Our current optical imaging results indicate that only a small fraction of synapses (~20 %) support spontaneous or evoked transmission at the expense of the other. However, it is likely that this fraction is higher than our estimates due to the inherent bias in fluorescent puncta selection. Accordingly, the model presented in Figure 8 is consistent with the proposal that some small synapses (<0.2 μm2) may indeed sustain solely evoked or spontaneous release. Second, the selection of fluorescent puncta that correspond to active synapses using 30 Hz stimulation may bias our results against a population of synapses that may show low levels of spontaneous release without significant evoked release. Nevertheless, our optical analysis is consistent with an earlier study in the frog neuromuscular junction, which found that the level of spontaneous release is relatively uniform across active zones and the location of spontaneous release corresponded well with the sites of evoked release, although the propensity of evoked release varied widely among active zones (Zefirov et al., 1995). Taken together with these earlier findings in hippocampal synapses and the frog neuromuscular junction, our results support the premise that spontaneous and evoked release have substantial overlap in their sites of origin, but they do not possess significant correlation with respect to their kinetics. Therefore, if most spontaneous and evoked release events originate from the same synapse then it can still be meaningful to record frequency of mEPSCs or mIPSCs to determine whether there is a loss or an increase in the number of synapses. However, it may be difficult to correlate this parameter with evoked release probability.

Our findings may also be accounted for by potential differences between fusion pore kinetics or glutamate release profile of spontaneous and evoked fusion events. For instance, in a given synapse, evoked fusion events may reach a higher percentage of receptors whereas spontaneous fusion events may activate only a small number of receptors (Cull-Candy and Leszkiewicz, 2004) although the two receptor populations overlap. This possibility contradicts several earlier observations. Both forms of fusion have been shown to equally stimulate AMPA receptors (Sun et al., 2002), which have approximately 100-fold less affinity for glutamate than NMDA receptors suggesting that they both can activate a number of receptors albeit below saturating levels (Mainen et al., 1999). In addition, this scenario is hard to reconcile with the mirror experiments presented in Figures 2, ,3,3, ,44 and and55 of this study, namely block of evoked or spontaneous fusion events leads to only limited occlusion of each other irrespective of the order at which they were blocked by MK-801.

A third proposal suggests that spontaneous fusion events may occur ectopically (Matsui and Jahr, 2003; Coggan et al., 2005), outside the active zones, as proposed by some earlier work (Colmeus et al., 1982). Our findings may partly support this possibility as long as this “ectopic” release occurs at discrete spots and activates a clustered set of adjacent receptors. The fact that the kinetics of spontaneous and evoked quantal events match under most circumstances (Diamond and Jahr, 1995; Isaacson and Walmsley, 1995; Van der Kloot, 1996; Wall and Usowicz, 1998; Sun et al., 2002) makes a diffuse form of ectopic release an unlikely option to account for our observations. Furthermore, the rapidity of MK-801 block of NMDA-mEPSCs is consistent with the premise that spontaneous fusion events occur in discrete sites thus repetitively activating a cluster of receptors rather than fusing at sites diffusely distributed along an axon.

The last possibility is that evoked and spontaneous fusion sites are compartmentalized within a single synapse presumably in the vicinity of a given active zone thus activating receptors in different subdomains of the PSD. We think this last model brings together the “different synapses” and “ectopic release” models in one scheme that could account for our data as well as earlier observations (Townsend et al., 2003). This proposal is also supported by the quantitative model we presented in Figure 8, which suggest that medium to large (>0.2 μm2 area) synapses can easily accommodate independent signaling via spontaneous and evoked release with some geometric constraints. However, our data does not exclude the possibility that small synapses (<0.2 μm2 area), which may be below the resolution of our optical experiments, can support spontaneous or evoked release exclusively and contribute to our electrophysiological observations. Accordingly, previous work showed that single vesicle fusion events activate only a small number of NMDA receptors (~3) that typically comprise less than 40% of the total number of NMDA receptors per postsynaptic site (Nimchinsky et al., 2004). Therefore, we think there is sufficient latitude for non-overlapping activation of NMDA receptors within a single synapse by evoked and spontaneous release events.

Probing the properties of asynchronous release

The findings discussed above gave us an opportunity to address a key question on the role of synaptotagmin 1 in controlling neurotransmitter release. Taking advantage of the differential activation of NMDA receptors by spontaneous and evoked release events, we could show that asynchronous release events still maintained the properties of synchronous evoked transmission by activating a set of NMDA receptors distinct from spontaneous events. In the absence of synaptotagmin 1, spontaneous release rate was significantly increased. Thus the increase in spontaneous release and loss of release synchrony seen in syt1 deficient synapses are separable phenotypes suggesting a dual role for synaptotagmin 1 in regulation of fusion. Furthermore, the asynchronous release elicited in the presence of Sr2+ was also selective in its ability to activate a set of NMDA receptors distinct from spontaneous events and shared with Ca2+-evoked release. Therefore, our results support the premise that asynchronous unitary events detected in Sr2+ provide a more accurate picture for the quantal properties of evoked release (Oliet et al., 1996).

Implications for neuronal signaling

In addition to their implications for the analysis of unitary neurotransmission, the findings we present here suggest a potential divergence in signaling triggered by evoked versus spontaneous activation of postsynaptic neurotransmitter receptors. Although, here, we did not detect a significant difference between the compositions of NMDA receptors activated by the two forms of release, this observation does not exclude differences in the downstream events triggered the two sets of NMDA receptors. In future experiments, it will be important to test whether other postsynaptic receptor types that respond to different neurotransmitters follow the same premise. In addition, it will be critical to examine the structural determinants of this putative functional compartmentalization within synapses and also investigate whether differential activation of receptors with spontaneous and evoked forms of fusion leads to activation of distinct signaling cascades in target neurons (Sutton et al., 2007).

Supplementary Material

Supp1

Acknowledgments

We thank K. Huber, H. Krämer, L. Monteggia, T. C. Südhof, J. Sun, D.G.R. Tervo and members of the Kavalali laboratory for helpful discussions, and for critically reading the manuscript. We are grateful to J. Xu and Z. Pang for sharing cultures from synaptotagmin-1 deficient mice. We thank Y. Zhu, and C.F. Stevens for the gift of synaptophysin-pHluorin construct. This work was supported by grants from the National Institute of Mental Health to E.T.K. D.A. is in part supported by a grant to Dr. T. C. Südhof from the National Institute of Mental Health (MH052804). KM is supported by a grant from National Institute of Drug Abuse (DA018109). E.T.K. is an Established Investigator of the American Heart Association.

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