Synaptic plasticity is widely believed to constitute a key mechanism for modifying functional properties of neuronal networks. This belief implicitly implies, however, that synapses, when not driven to change their characteristics by physiologically relevant stimuli, will maintain these characteristics over time. How tenacious are synapses over behaviorally relevant time scales? To begin to address this question, we developed a system for continuously imaging the structural dynamics of individual synapses over many days, while recording network activity in the same preparations. We found that in spontaneously active networks, distributions of synaptic sizes were generally stable over days. Following individual synapses revealed, however, that the apparently static distributions were actually steady states of synapses exhibiting continual and extensive remodeling. In active networks, large synapses tended to grow smaller, whereas small synapses tended to grow larger, mainly during periods of particularly synchronous activity. Suppression of network activity only mildly affected the magnitude of synaptic remodeling, but dependence on synaptic size was lost, leading to the broadening of synaptic size distributions and increases in mean synaptic size. From the perspective of individual neurons, activity drove changes in the relative sizes of their excitatory inputs, but such changes continued, albeit at lower rates, even when network activity was blocked. Our findings show that activity strongly drives synaptic remodeling, but they also show that significant remodeling occurs spontaneously. Whereas such spontaneous remodeling provides an explanation for “synaptic homeostasis” like processes, it also raises significant questions concerning the reliability of individual synapses as sites for persistently modifying network function.

Neurons communicate via synapses, and it is believed that activity-dependent modifications to synaptic connections—synaptic plasticity—is a fundamental mechanism for stably altering the function of neuronal networks. This belief implies that synapses, when not driven to change their properties by physiologically relevant stimuli, should preserve their individual properties over time. Otherwise, physiologically relevant modifications to network function would be gradually lost or become inseparable from stochastically occurring changes in the network. So do synapses actually preserve their properties over behaviorally relevant time scales? To begin to address this question, we examined the structural dynamics of individual postsynaptic densities for several days, while recording and manipulating network activity levels in the same networks. We found that as expected in highly active networks, individual synapses undergo continual and extensive remodeling over time scales of many hours to days. However, we also observed, that synaptic remodeling continues at very significant rates even when network activity is completely blocked. Our findings thus indicate that the capacity of synapses to preserve their specific properties might be more limited than previously thought, raising intriguing questions about the long-term reliability of individual synapses.

To evaluate the tenacity of individual synaptic structures over behaviorally relevant time scales and differentiate between activity-dependent and activity independent-synaptic remodeling, an experimental system is needed in which both structural dynamics of individual synapses and electrical activity can be monitored continuously and simultaneously at sufficiently high temporal resolutions for very long periods. At present, this is an extremely challenging requirement, in particular where in vivo studies are concerned. We therefore developed a novel system, based on networks of rat cortical neurons in primary culture, that allowed us to continuously follow and record the structural dynamics of individual PSDs over time scales of minutes to weeks while concomitantly recording (and manipulating) network activity in the same preparations. We find that the vast majority of PSDs in this preparation undergo significant, continuous remodeling over time scales of many hours and days. The direction and extent of PSD remodeling are strongly affected by network activity levels, but remodeling does not cease upon suppression or elimination of activity. Our findings, described below, thus indicate that the tenacity exhibited by individual synapses over time scales of days is rather limited and may indicate that structural (and by extension, functional) properties of individual synapses experience significant drift over long durations.

In most of the aforementioned studies, synapses were visualized by means of volume-filling fluorescent dyes (mainly enhanced green fluorescent protein [EGFP] or its spectral variants) and identified on the basis of typical pre- and postsynaptic morphological features (i.e., axonal varicosities and dendritic spines, respectively), whereas functionally relevant reporters, such as synaptic vesicle, postsynaptic receptor, active zone, or postsynaptic density (PSD) molecules were rarely used. Furthermore, even though manipulations aimed at altering network activity were performed in some of these studies, actual network activity was not recorded. Thus, the actual relationships between synaptic tenacity, synaptic remodeling, and network activity over these long time scales remained unknown.

Despite the molecular dynamics of synaptic constituents, most central nervous system (CNS) synapses appear to be quite persistent, although some degree of synapse formation and elimination is observed, depending on brain region, type of synapse, animal age, and imaging techniques [13] – [20] (reviewed in [8] , [21] ). Interestingly, however, even persistent synapses, when examined over long time scales (days), seem to exhibit considerable morphological changes (for example, [14] , [15] , [17] , [22] , [23] ; see also [18] ). In most of these studies, it was surmised that the observed changes in synaptic morphology represented structural manifestations of synaptic plasticity processes.

The advent of molecular imaging techniques and the ability to study the molecular dynamics of specific molecules are gradually leading to the realization that synapses are not static, rigid structures; rather, they are made of multimolecular protein ensembles that exhibit significant dynamics at time scales of seconds to hours. Such dynamics include the recruitment and dispersal of regulatory constituents, lateral diffusion, endocytosis and exocytosis of postsynaptic neurotransmitter receptors, cytoskeletal dynamics and spine “morphing”, loss, incorporation, and turnover of scaffold molecules, and the interchange of synaptic molecules, multimolecular complexes, and synaptic vesicles among neighboring synapses (reviewed in [3] – [11] ). When considering the bewildering dynamics exhibited by synaptic molecules, it becomes apparent that the long-term tenacity of synaptic structure and, by extension, synaptic function is not at all an obvious outcome. Yet to date, very little is known on the long-term tenacity of individual synapses [12] .

Synapses are widely believed to constitute key loci for modifying the functional properties of neuronal networks, possibly providing the basis for phenomena collectively referred to as learning and memory [1] , [2] . Indeed, an overwhelming body of literature supports the notion that synapses are “plastic”, that is, change their functional characteristics in response to specific activation patterns. The hypothesis that activity-dependent changes to synaptic characteristics constitutes a key mechanism for modifying neuronal network function also implies, however, that synapses, when not driven to change their characteristics by physiologically relevant stimuli, should retain these characteristics over time. Otherwise, physiologically relevant modifications to network function would be gradually lost due to stochastic, spurious changes or spontaneous drift. Thus, it might be expected that the capacity of synapses for directed change—synaptic plasticity—should be accompanied by a tendency to retain their characteristics at all other times, a phenomenon we will refer to here as “synaptic tenacity”.

Results

Spontaneous Changes in Network Activity, PSD Number, and PSD Size We initially set out to the examine the long-term stability of PSD-95:GFP puncta under baseline conditions, that is, without experimentally manipulating network activity. To that end, we used preparations maintained in culture for at least 17 d. At this stage, networks of rat cortical neurons are considered to be relatively mature, and beyond the phase of extensive dendrite extension and synapse formation. The preparations were mounted on the CLSM and followed by combined time-lapse imaging and electrophysiological recordings as described above. At the end of each experiment, spike counts per electrode were extracted from the electrophysiological recordings, and the numbers of PSD-95:GFP puncta and their respective fluorescence intensities were extracted from maximal intensity projections of Z-stacks for each time point and each site (see Materials and Methods). These experiments resulted in two highly consistent observations. The first concerns network activity. As noted above, cultured networks of dissociated cortical neurons typically develop complex patterns of spontaneous activity composed of asynchronous action potentials and synchronized bursts (Figure 2A–2C). These forms of activity were observed here as well, as soon as recording was initiated. However, over the first 1–2 d, we consistently observed significant elevations of spontaneous activity levels (Figure 3A). These elevations reflected both increases in the number of “active” electrodes as well as increases in the frequencies of action potentials measured from individual electrodes (Figure S3). Intermittently, periods of “superbursts” (i.e., bursts of bursts; [49]) were recorded (see below), giving rise to significant variability in action potential counts from one minute to the next. This gradual increase in network activity at the beginning of the experiment was observed in practically all experiments regardless of preparation age, indicating that it was due, somehow, to the environmental conditions introduced during the experiments (see below). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Evolution of network activity and PSD-95:GFP puncta population properties. (A) Spontaneous activity recorded for 93 h from a network of cortical neurons growing on a MEA dish, starting with the mounting of the preparation on the combined MEA recording/imaging system. Activity is expressed as action potentials (measured from all electrodes) per second. (B) Changes in the number of discernable PSD-95:GFP puncta in the same preparation during the same recording period (eight neurons). (C) Normalized distribution of fluorescence intensities of all discernable PSD-95:GFP puncta at each time point (bin size = 50 gray-scale units). Each vertical line represents the fractional distribution of fluorescence intensities for that time point, color coded according to scale bar in (G). Mean puncta fluorescence is shown as a black line. (D) Four representative histograms (bin size = 50 gray-scale units) from the time points marked and color coded by arrows in (C). Note that the initial broadening of the distribution of PSD-95:GFP puncta fluorescence intensities is followed by a constriction of this distribution. Also note that the increased bursting activity starting at approximately 25 h (manifested in the increased variability in spike rates from one minute to the other) is associated with an increase in PSD-95:GFP puncta numbers. (E) A similar experiment to that shown in (A–D), except that here, TTX was added (arrow) to the MEA dish (and to the perfusion medium) an hour after mounting the preparation. (F) Changes in the number of discernable PSD-95:GFP puncta (five neurons). (G) Normalized distribution of fluorescence intensities of all discernable PSD-95:GFP puncta at each time point. (H) Four representative histograms from the time points marked and color coded by arrows in (G). Unlike the experiment shown in (A–D), the broadening of the PSD-95:GFP puncta fluorescence intensity distribution was not reversed. https://doi.org/10.1371/journal.pbio.1000136.g003 The second consistent observation concerns the morphological complexity of postsynaptic structures along dendritic segments. In practically all experiments, we observed a gradual increase in the total number of PSD-95:GFP puncta, mainly (but not exclusively) due to increased density of PSD-95:GFP puncta along existing dendritic segments, at both spine tips and shafts (Figures 1C and 3B). Moreover, the population of PSD-95:GFP puncta changed from one that was relatively uniform in terms of fluorescence intensity to one that contained both very large, bright puncta as well as many small, dim puncta (Figures 1C, 3C, and 3D). Interestingly, the broadening of the puncta intensity distribution was usually transient, and was partially reversed when network activity reached relatively high levels after 1–2 d (Figure 3A, 3C, and 3D). As the majority of PSD-95:GFP puncta were typically juxtaposed against functional presynaptic boutons (Figure S1; see also [35]), the observed increases in PSD-95:GFP puncta number reflected, in all likelihood, increased numbers of glutamatergic synapses. Furthermore, given that PSD size, PSD-95:GFP fluorescence, spine head dimensions, AMPA receptor number, and glutamate-induced synaptic currents, are well correlated [16],[50]–[54] changes in PSD-95:GFP content, probably reflected changes in PSD size and possibly in synaptic strength [8],[53]. The (post)synaptic remodeling described above resulted in dendrites assuming morphological characteristics more akin to those of dendrites in vivo. Yet we could not rule out the possibility that these morphological changes were actually reflecting pathological processes induced by the environmental conditions during experiments or damage inflicted by continuous imaging. To examine the possibility that the experimental conditions were detrimental to neuronal vitality, we used the same system to follow the development of less mature networks in which vigorous growth and synapse formation are known to occur, because here, pathological phenomena such as growth cessation, axon/dendrite retraction, and synapse elimination, are clearly recognizable. To that end, preparations were mounted on the CLSM starting from day 9–10 in vitro, maintained in the environmental conditions described above, and imaged at higher frequencies (every 10 min instead of 30) for about 1 wk. Dendritic development in these experiments appeared to proceed as expected: new branches were added, synapses were formed at high rates, and network activity levels increased 10- to 20-fold (Figure S4 and Video S1). In none of these experiments (n = 7) did we observe signs of damage. In fact, these experiments resulted in exciting and, to the best of our knowledge, unprecedented recordings of dendritic development and synapse formation that will be described elsewhere. These experiments, therefore, do not support the possibility that the experimental conditions used here adversely affect neuronal viability, and lead us to conclude that the synaptic remodeling described above is not secondary to pathological processes.

Relationships between Network Activity and the Population Distribution of PSD Size In the aforementioned experiments, we observed significant changes in PSD-95:GFP puncta number and fluorescence intensity, which, in all likelihood, reflected changes in glutamatergic synapse number and PSD size. This remodeling occurred concomitantly with significant changes in network activity, which pointed to the possibility that the two phenomena might be causally related. It should be noted, however, that unlike network activity, that generally increased over time (Figure 3A), the initial broadening of the PSD-95:GFP fluorescence intensity distribution (and the gradual increase in mean puncta fluorescence) was usually followed by a second phase during which the intensity distribution partially recovered (as did mean puncta fluorescence; Figure 3C and 3D) and thereafter remained stable for days, indicating that the potential relationships between the two phenomena are not straightforward. To determine whether the observed changes in PSD-95:GFP puncta number and fluorescence intensity were dependent on changes in network activity, we repeated the experiments described above except that here, spontaneous network activity was blocked by adding tetrodotoxin (TTX) about an hour after the experiments were started. As shown in Figure 3E–3H, blocking network activity did not block the initial broadening of the PSD-95:GFP fluorescence intensity distribution. However, the second phase (the partial recovery of the intensity distribution) was completely lost. Instead, the distributions of PSD-95:GFP puncta fluorescence intensities continued to broaden, and mean puncta fluorescence continued to increase (Figure 3G and 3H). In addition, the number of puncta did not increase over time, and in fact, gradual decreases in puncta numbers were observed (Figure 3F). These experiments indicate that the initial broadening of PSD-95:GFP puncta fluorescence intensity distribution is not driven by activity. Rather, it seems to be driven by the exposure to environmental conditions during experiments. Given that ambient temperature and atmospheric conditions were identical to those in the incubators in which preparations were maintained, the most likely “culprit” is the slow perfusion. Indeed, these phenomena are not observed if perfusion is not applied (unpublished data). On the other hand, in the absence of perfusion, the long-term viability of these preparations was drastically impaired. Interestingly, media turnover rates (∼0.15%/min) were one to two orders of magnitudes lower than cerebrospinal fluid (CSF) turnover rates in the intact rat brain (1% to 16%/min; [55]), indicating that perfusion rates were not excessively high. In contrast to the initial broadening of PSD-95:GFP fluorescence intensity distributions, the subsequent constriction of fluorescence intensity distributions was clearly dependent on network activity. This dependence indicates that an increase in activity levels is associated with a reduction in mean PSD size. This finding is consistent with the concept of “synaptic scaling” [56]–[58], that is, the adjustment of synaptic strength to match neuronal activation levels. Interestingly, following the initial broadening and subsequent constriction, PSD-95:GFP puncta fluorescence intensity distributions remained relatively stable as long as activity levels did not change significantly (as exemplified in Figure 3C). These observations are consistent with the possibility that PSD size is generally stable, with changes in activity followed by uniform and gradual scaling of PSD size (multiplication by a scalar, for example). However, as shown next, this does not seem to be the case.

Modeling Relationships between Network Activity and Synaptic Remodeling The long-term recordings of synaptic remodeling described so far indicate that (1) spontaneous network activity maintains distributions of synaptic sizes within rather constrained boundaries; (2) reductions in network activity levels result in a broadening of synaptic size distributions, increases in mean synaptic size, and reductions in synapse numbers; (3) most synapses exhibit significant changes in size over time; (4) in active networks, changes in synaptic size are partially dependent on momentary synapse size: large synapses tend to become smaller, whereas small synapses tend to become larger; and (5) when activity is blocked or significantly suppressed, synapses continue to change their sizes, but the direction and extent of these changes become independent of momentary synapse size. We hypothesized that the phenomenological relationships between network activity and synaptic remodeling described above could be explained by the following set of rules: (1) synapses continuously undergo spontaneous, activity-independent changes (drift) in their size; (2) activity acts to reduce the size of large synapses on the one hand, and increase the size of small synapses on the other; (3) new, small synapses are continually formed at a constant rate; and 4) synapses whose size is reduced beneath some threshold are eliminated. To examine whether this set of rules could, at least in principle, explain the phenomena described above and produce synaptic size distributions similar to those measured experimentally, we created a simple numerical model in which sizes and fates of individual synapses were updated over time according to these four rules (see legend of Figure 7 and Materials and Methods for further details). Figure 7C–7F shows simulations seeded with the initial puncta counts and intensities measured in the experiments of Figures 4 and 5. As these figures show, the numerical simulations recapitulated the major phenomenological relationships between network activity and synaptic size distributions (Figure 7C and 7E). Furthermore, they predicted correctly the changes in synaptic counts measured in these experiments (Figure 7D and 7F). These findings indicate that relationships between synaptic size distributions and network activity levels can be accounted for, at least in principle, by the four simple rules described above without a need for explicit “scaling” or compensatory mechanisms invoked by reductions in network activity levels (although this does not preclude their existence). Although the simulated data generally approximated the experimental measurements quite well, they failed to account for a small population of particularly large synapses observed in active networks (visible as blue dots in the upper regions of the histograms of Figure 7C and 7E). We do not think this is related to imperfect simulation parameters but rather to the existence of at least one other “rule” that drives the formation of large synapses in highly active networks. Indeed, we noted that relatively large synapses tended to appear during periods of particularly high levels of synchronous activity, often associated with “superbursting” [48], observable as periods of significant variability in action potential counts from one minute to the other. In fact, a comparison of particularly bright (1.5 standard deviations above mean puncta fluorescence) PSD-95:GFP puncta appearance rates to burst rates revealed strong temporal relationships between these two phenomena (Figure S6A), relationships observed in practically all experiments in which superbursting occurred. In one striking example, tracking particularly bright puncta backward in time revealed that 18 out of 18 such bright puncta exhibited dramatic increases in fluorescence with the onset of superbursting or seemed to appear de novo (Figure S6B, S6C, and S6E; see also Figure 6, puncta 1 and 4). It is likely that these large PSD-95:GFP puncta represent synapses that underwent forms of potentiation associated with spine-head enlargement (e.g., [23],[53],[59]–[64]). In fact, we suspect that high levels of synchronized activity, rather than high activity levels per se are also the driving force behind the aforementioned tendency of small puncta to grow larger in highly active networks (see [65] and discussion below). As we did not have access to the precise firing timings of individual pre- and postsynaptic partners, this possibility remains somewhat speculative. Interestingly, however, as our system did allow us to follow these synapses for relatively long periods, we were able to compare their tenacity to that of the rest of the synaptic population. These observations indicated that newly enlarged synapses did not necessarily fair much better than other synapses in terms of their long-term tenacity, even in the presence of TTX (Figure S7; see also [23],[59]), although their lifespans were typically longer (unpublished data).