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 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 , , and 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 , 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