Introduction

One of the most studied phenomena triggered by prolonged activity blockade is the increase in amplitudes of miniature excitatory postsynaptic currents (mEPSCs) in neurons. First demonstrated in cultures of dissociated cortical neurons (Turrigiano et al., 1998) and spinal cord neurons (O’Brien et al., 1998), the compensatory response to prolonged loss of activity has been dubbed “homeostatic synaptic plasticity” (Turrigiano and Nelson, 2004; Pozo and Goda, 2010). These first two studies provided evidence that glutamate receptors were also increased after prolonged inactivity, via an increased response to exogenously applied glutamate (Turrigiano et al., 1998) and increased immunofluorescent labeling of GluA1 receptors at synaptic sites (O’Brien et al., 1998). The accompanying increase in synaptic AMPA receptors has been confirmed with immunofluorescence in multiple studies of hippocampal and cortical cultures treated with TTX, including (Wierenga et al., 2005; Hou et al., 2008; Ibata et al., 2008; Jakawich et al., 2010; Tatavarty et al., 2013; Xu and Pozzo-Miller, 2017; Dubes et al., 2022). It is now well accepted that the homeostatic increase in mEPSC amplitudes in neurons is due to an increase in postsynaptic AMPA receptors.

In our previous work, we found that a TTX cuff applied for 48 hrs around the sciatic nerve in mice led to an increase in the amplitude of the miniature endplate currents (mEPCs) recorded in tibialis anterior muscles. Surprisingly, and unlike at central synapses, we could find no evidence that the acetylcholine receptors levels at the neuromuscular junction (NMJ) were increased in the TTX-cuff treated mice (Wang et al., 2005). This result led us to search for presynaptic molecules that might homeostatically regulate the presynaptic quantum. In previous studies in chromaffin cells, we identified the small GTPase Rab3A, a presynaptic vesicle protein, as a regulator of synaptic vesicle fusion pore opening (Wang et al., 2008), so we examined whether deletion of Rab3A (Rab3A^-/-) might prevent homeostatic upregulation of mEPC amplitude at the NMJ. The results were clear: the homeostatic increase in mEPC amplitude induced by the TTX cuff was strongly reduced in the Rab3A^-/- mouse, and was completely abolished in the Earlybird mutant (Rab3A^Ebd/Ebd), which has a single point mutation in Rab3A that causes a shift towards early awakening due to a shorter circadian period, that is more dramatic than that of the Rab3A deletion mouse (Kapfhamer et al., 2002; Wang et al., 2011). These results led us to conclude that the homeostatic increase in mEPC amplitude at the NMJ is a presynaptic phenomenon.

It has remained somewhat of a mystery what the role of Rab3A is in synaptic transmission. The Rab3A^-/- mouse has minimal phenotypic abnormalities, with evoked synaptic transmission and mEPSCs essentially normal in hippocampal slices (Geppert et al., 1994). At the Rab3A^-/- NMJ, reductions in evoked transmission were detected, but only under conditions of reduced extracellular calcium (Coleman et al., 2007). There are some modest changes in short term plasticity during repetitive stimulus trains, with increased depression observed in response to moderate frequencies in hippocampal slices (Geppert et al., 1994) and at the mammalian NMJ (Coleman and Bykhovskaia, 2009), but increased facilitation in response to high frequency stimulation at the mammalian NMJ (Coleman and Bykhovskaia, 2010). The most dramatic effect of loss of Rab3A is the abolishment of a presynaptic form of long-term potentiation (LTP) at the mossy fiber-CA3 synapse that is induced by 25 Hz stimulation in the presence of NMDA blockers (Weisskopf et al., 1994; Castillo et al., 1997). Our result that prolonged inactivity-induced plasticity of mEPC amplitude at the mammalian NMJ is disrupted with loss of function of Rab3A further cements a role for Rab3A in activity-dependent plasticity of synaptic strength. However, a crucial question that remains is whether the TTX-cuff induced effect on the amplitude of the spontaneous synaptic current at the NMJ shares underlying mechanisms with central synapses. We set out to answer this question for the more well-studied phenomenon of homeostatic plasticity of mEPSCs in dissociated mouse cortical neurons prepared from wild type, Rab3A^-/- mice and Rab3a^Ebd/Ebd mice treated with TTX for 48 hrs. We show here that homeostatic synaptic plasticity of mEPSC amplitude was strongly reduced in cultures from Rab3A^-/- mice, and completely abolished in cultures from Rab3A^Ebd/Ebd mice, supporting a shared mechanism with the neuromuscular junction.

Results

We previously reported that mixed cultures of cortical neurons and glia prepared from postnatal day 0-2 mouse pups responded to a block of action potential-mediated activity by a 48 hr TTX treatment with an increase in mEPSC amplitude (Hanes et al., 2020). Here, we asked the question whether cortical cultures prepared from mice lacking the small GTPase Rab3A, or, expressing a point mutation of Rab3A, Rab3A Earlybird, have an altered homeostatic plasticity response to a prolonged loss of network activity. The Rab3A Earlybird mutation was discovered in a mutagenesis screen in mice for genes involved in the rest-activity cycle (Kapfhamer et al., 2002). In that study, the authors concluded that the Earlybird mutation was likely dominant-negative, as the shift in circadian period was substantially more dramatic than that observed in the Rab3A^-/- mice. As described in the Introduction, the effects of loss of Rab3A have been modest, so we included experiments using the Rab3A Earlybird mutant mice to see if there would be a more robust phenotype, and in doing so, strengthen our conclusions based on the Rab3A deletion mice. To obtain Rab3A^-/- and Rab3A^Ebd/Ebdhomozygotes, we established two mouse colonies of heterozygous breeders with cultures prepared from pups derived from a final breeding pair of homozygotes. Although we backcrossed Rab3A^+/- with Rab3A^+/Ebd for 11 generations, clear differences in mEPSC amplitudes in untreated cultures (see below) and in calcium current amplitudes in adrenal chromaffin cells (unpublished obs.) remained. Therefore, throughout this study we keep the two strains separate and there are two Rab3A^+/+ or ‘wild type’ phenotypes.

We found that loss of Rab3A resulted in a greatly impaired homeostatic increase after activity blockade. Example current traces of spontaneously occurring mEPSCs recorded from pyramidal neurons in untreated (CON) 13-14 DIV cortical cultures and sister cultures treated with 500 nM TTX for 48 hr prepared from wild type and Rab3A^-/- mice in the Rab3A^+/- colony are shown in Figures 1A and B, respectively. Average mEPSC waveforms from the same recordings are shown in Figures 1C and D. The mean mEPSC amplitudes for 30 control and 23 TTX-treated neurons from Rab3A^+/+ cultures are displayed in the box and whisker plot in Figure 1E; after activity blockade the average mEPSC amplitude increased from 13.9 ± 0.7 pA to 18.2 ± 0.9 pA. In contrast, in cultures prepared from Rab3A^-/- mice, the average mEPSC amplitude was not significantly increased, for 25 untreated cells and 26 TTX-treated cells (Figure 1F, 13.6 ± 0.1 pA vs. 14.3 ± 0.6 pA). We found that TTX treatment also resulted in an increase in mEPSC frequency in cultures prepared from Rab3A^+/+ mice, as shown in Figure 1G (CON, 2.26 ± 0.37 sec^-1; TTX, 4.62 ± 0.74 sec^-1). This TTX-induced frequency increase was strongly reduced in cultures from Rab3A^-/- mice, but there were a few outliers with very high frequencies still present after TTX treatment (Figure 1H, CON, 2.74 ± 0.49 Hz; TTX, 3.23 ± 0.93 Hz).

Figure 1.

We next examined homeostatic plasticity in cultures prepared from wild type mice in the Rab3A^+/Ebd colony. Example current traces of mEPSCs, and average mEPSC waveforms, are shown in Figures 2A and C, respectively, for cortical cultures prepared from wild type mice in the Rab3A^+/Ebd colony. Treatment with TTX for 48 hr led to a significant increase in the average mEPSC amplitude of 23 TTX-treated cells when compared to 20 untreated cells (Figure 2E, CON, 11.0 ± 0.6 pA; TTX, 15.0 ± 1.3 pA). We note here that while the two wild type strains respond very similarly to activity blockade, the mean mEPSC amplitude in untreated cultures was significantly different in the two wild type strains, 13.9 ± 0.7 pA, wild type from Rab3A^+/- colony; 11.0 ± 0.6 pA, wild type from Rab3A^+/Ebd colony (p = 0.004, Kruskal-Wallis test).

Figure 2.

We found a complete disruption of homeostatic plasticity in cortical cultures prepared from Rab3A^Ebd/Ebd mice, as can been seen in viewing example mEPSC traces and average mEPSC waveforms (Figures 2B and D, respectively). The lack of TTX effect was confirmed in a comparison of mEPSC amplitude means for 21 untreated and 22 TTX-treated cells (Figure 2F, CON, 15.1 ± 1.0 pA vs. TTX, 14.6 ± 1.1 pA). There was a trend towards increased mEPSC frequency in TTX-treated cultures from this wild type strain, but the difference was not as robust as for the wild type cultures from Rab3A^+/- colony, and did not reach statistical significance (Figure 2G, CON, 1.15 ± 0.19 sec^-1; TTX, 2.54 ± 0.55 sec^-1). This trend was not observed in neurons from cultures prepared from Rab3A^Ebd/Ebd mice, likely due to an increase in frequency in untreated cells—frequencies remained high in TTX-treated cells (Figure 2H, CON, 1.71 ± 0.41 sec^-1; TTX, 3.05 ± 0.80 sec^-1). Our results show that the homeostatic increase in mEPSC amplitude after activity blockade is disrupted in both Rab3A^-/- and the Rab3A^Ebd/Ebdcortical neurons, strongly supporting a crucial role of functioning Rab3A in this process.

The disruption in homeostatic plasticity differs for Rab3A^-/- and Rab3A^Ebd/Ebd. In the Rab3A^-/- data set, mEPSCs from untreated cultures were indistinguishable from mEPSCs from Rab3A^+/+ untreated cultures, demonstrating loss of Rab3A has no impact on basal activity mEPSC amplitudes. In the Rab3A^Ebd/Ebd data set, mEPSC amplitudes from untreated cultures were significantly larger than those of untreated cultures from wild type mice, as can be seen comparing the CON values in Figures 2E and F (CON, Rab3A^+/+, 11.0 ± 0.7 pA, vs. CON, Rab3A^Ebd/Ebd, 15.1 ± 1.0 pA, p = 0.0027, Kruskal-Wallis test). The increase in mEPSC amplitude in cultures from Rab3A^Ebd/Ebd mice is consistent with the increase in mEPC amplitude we observed at the Rab3A^Ebd/EbdNMJ (Wang et al., 2011).

The small GTPase Rab3A is generally thought to function presynaptically to regulate synaptic vesicle trafficking, possibly in an activity-dependent manner (Castillo et al., 1997; Lonart et al., 1998; Leenders et al., 2001; Schluter et al., 2006; Coleman and Bykhovskaia, 2009; Tian et al., 2012). In contrast, homeostatic plasticity of mEPSC amplitude has been attributed to an increase in postsynaptic receptors on the surface of the dendrite (O’Brien et al., 1998; Turrigiano et al., 1998). At the NMJ in vivo, we could find no evidence for an increase in AChRs after TTX block of the sciatic nerve in vivo (Wang et al., 2005). Is Rab3A required for the homeostatic increase in surface AMPA-type glutamate receptors that has been confirmed by multiple studies (see Introduction)?

We explored whether Rab3A might be acting to increase postsynaptic AMPA receptors. In some studies of homeostatic synaptic plasticity it has been found that application of a Ca²⁺- permeable AMPA receptor-specific inhibitor reversed the homeostatic increase in mEPSC amplitude (Ju et al., 2004; Thiagarajan et al., 2005; Sutton et al., 2006). Figure 3A shows that the TTX-induced increase in mean mEPSC amplitude was nearly identical in a set of 11 CON and 11 TTX-treated cells before and after treatment with 1-Naphthyl acetyl spermine trihydrochloride (NASPM), a synthetic analogue of the Ca²⁺-permeable AMPA receptor inhibitor philanthotoxin (before NASPM, CON 12.9 ± 3.5 pA; TTX, 17.5 ± 3.1 pA; after NASPM, CON 11.9 ± 2.6 pA; TTX 16.1 ± 3.5 pA). This result indicates that the TTX-induced increase in mEPSC amplitude does not depend on Ca²⁺-permeable receptors, since the effect of TTX clearly remained when their presence was removed by acute NASPM application. We do not think that there was any technical issue with the NASPM application, because overall, mEPSC amplitudes were reduced modestly in both untreated and TTX-treated cultures (Figure 3B, CON, before NASPM, 12.9 ± 3.5 pA; after NASPM, 11.9 ± 2.6 pA; TTX, before NASPM, 17.5 ± 3.1 pA; after NASPM, 16.1 ± 3.5 pA). Furthermore, NASPM consistently reduced mEPSC frequency (Figure 3C, CON, before NASPM, 1.84 ± 0.55 sec^-1; after NASPM, 1.56 ± 0.53 sec^-1; TTX, before NASPM, 4.40 ± 3.51 sec^-1; after NASPM, 2.68 ± 2.25 sec^-1). Similar to the data in Figure 1, frequency is significantly increased after TTX treatment (p = 0.04), but this difference is no longer significant after NASPM application (p = 0.20). One explanation for the effect on frequency is that there are synaptic sites that express only homomers of Ca²⁺-permeable AMPA receptors (GluA2-lacking AMPARs); mEPSCs from these sites would be expected to be completely blocked by NASPM (see cartoon description in Figure 3D, Left). We cannot rule out an alternative explanation, that presynaptic Ca²⁺-permeable AMPA receptors normally enhance release probability, and NAPSM prevents this action. The magnitude of the frequency reduction following acute NASPM appears to be greater after TTX treatment, suggesting that loss of activity could promote the establishment of synaptic sites that contain only Ca²⁺-permeable receptors. However, these new sites do not appear to contribute to the increase in mEPSC amplitude (Figure 3A). The lack of effect of NASPM rules out the contribution of Ca²⁺-permeable AMPA receptors GluA1, GluA3 and GluA4 (Hollmann et al., 1991; Burnashev et al., 1992; Jonas and Burnashev, 1995), as well as Ca²⁺-permeable forms of Kainate receptors GluA5 and GluA6 (Koike et al., 1997; Sun et al., 2009) to homeostatic synaptic plasticity.

Figure 3.

Having established that homomeric Ca²⁺-permeable receptors are not contributing to the homeostatic increase in mEPSC amplitude, we turned to immunofluorescence and confocal imaging to assess whether GluA2 receptor expression, which will identify GluA2 homomers and GluA2 heteromers, was increased in our wild type mouse cortical cultures following 48 hr treatment with TTX. Since mEPSCs necessarily report synaptic levels of receptors, we used VGLUT1-immunofluorescence to identify synapses on pyramidal primary apical dendrites labeled with MAP-2 immunofluorescence. We focused on the primary dendrite of pyramidal neurons as a way to reduce variability that might arise from being at widely ranging distances from the cell body, or, from inadvertently sampling dendritic regions arising from inhibitory neurons. In addition, it has been shown that the increase in receptors is most dramatic in this region (Turrigiano et al., 1998). Figure 4 (Top) shows two pyramidal neurons, one from an untreated culture on the left, and one from a TTX-treated culture on the right, prepared from Rab3A^+/+ mice. To measure characteristics of synaptically located GluA2 receptor clusters, we zoomed in on the primary dendrite (region in white rectangle). The zoomed regions for single confocal sections of the selected area are shown below and contain pairs of VGLUT1- and GluA2-immunofluorescent clusters indicated by white trapezoidal frames. A dendrite typically required ∼10 confocal sections to be fully captured, and the total number of synaptic pairs for all the sections imaged on a dendrite averaged 20 (20.4 ± 6.5 (SD); range, 11-38) so this is an atypically high number of pairs within a single section; these particular dendrites and sections were selected for illustration purposes. In addition to the synaptic pairs, we observed many GluA2-immunofluorescent clusters not associated with VGLUT1 immunofluorescence (a few are indicated with white arrows), most probably extrasynaptic receptors. There were also GluA2- positive clusters present outside of MAP2-positive dendrites, which may be located on astrocytes (Fan et al., 1999). Finally, GluA2 immunofluorescent clusters close to VGLUT1 immunofluorescence but not located along any apparent MAP2-positive neurites suggests the presence of axon-axonal contacts, although VGLUT1 has also been detected in astrocytes (Ormel et al., 2012). Only sites that contained both VGLUT1 and GluA2 immunofluorescence close to the primary MAP2-postive dendrite or a secondary branch along this primary dendrite were selected for analyses. In the experiment from which these images were collected, we analyzed the distance from the cell body for each synapse. The average distance from the cell body, for dendrites from the untreated cell, was 38.5 ± 2.8 μm; for the TTX-treated cell, it was 42.4 ± 3.2 μm (p = 0.35, Kruskal-Wallis test). Since the greatest distance from the cell body is set by the outer limit of the zoom window, and that window was placed adjacent to the cell body, distances in other experiments would be in this same range.

Figure 4.

Variability in the magnitude of the homeostatic response from culture to culture is averaged out in physiological experiments by the pooling of data from many cells across many cultures. To reduce the necessity for many cultures, we chose to pair experiments in the same cultures by recording mEPSCs from one set of coverslips, and processing another set of coverslips from the same culture for immunofluorescence. We also wanted to determine if the homeostatic response of mEPSC amplitudes varied from culture to culture, and if so, whether the GluA2 receptor levels varied in parallel. We completed this matched paradigm of physiology and immunofluorescence for 3 cultures prepared from Rab3A^+/+ mice. We first present the results as means for each culture, for control and TTX-treated dishes, for mEPSC data (Figure 5A) and imaging data (Figure 5B). Levels of GluA2 immunofluorescence at synaptic sites were quantified by the size of the GluA2-immunofluorescent receptor cluster, because this showed the greatest response to activity blockade; the average intensity value, and the integral of intensity values across the area of the cluster, are included in a summary of the data in Table 1. Three of three cortical cultures showed an increase in mean mEPSC amplitude after TTX treatment. In contrast, one of the same three cultures showed a decrease in mean GluA2 receptor cluster size. Therefore, the decrease in receptor levels in Culture #3 was due to something other than a lack of homeostatic effect on mEPSCs in that culture. Because there was a clear trend towards increased GluA2 receptor cluster size, but the receptor response appears more variable, we performed immunofluorescence measurements on two additional cortical cultures, both of which showed modest increases after TTX treatment (Figure 5C). We confirmed that there was not an increase in GluA1 receptor levels in two matched cultures that showed robust homeostatic increases in mEPSC amplitudes (Supplementary Figure 1A, culture mean mEPSC amplitudes; Supplementary Figure 1B, culture mean GluA1 receptor cluster sizes).

Figure 5.

Table 1.

We next pooled data from the multiple cultures to examine effects of activity blockade on GluA2 receptors at the level of cell means. Because the average number of synaptic sites identified in the primary dendrites in three cultures was 20.4, we also took 20 mEPSC samples from each cell, the 21^st to the 40^th mEPSC recorded, to minimize any differences in variability between GluA2 receptors and mEPSCs that might be due to a difference in sampling. In the data pooled from the 3 matched experiments, neurons from Rab3A^+/+ cultures showed a significant increase in mean mEPSC amplitudes following activity blockade (Figure 5D; Table 1). This result indicates that the homeostatic response averaged across the 3 cultures was very similar to, but slightly smaller than, that of the data set presented in Figure 1. The mean for size of GluA2 immunofluorescent receptor clusters showed a trend towards higher values after activity blockade (18.1%) that was of similar magnitude to the mEPSC amplitude increase (19.7%), but did not reach statistical significance for 3 cultures (Figure 5E) or 5 cultures (Figure 5F; Table 1). As expected, mean mEPSC amplitude was not increased following activity blockade in the data pooled from a new set of 3 Rab3A^-/- cultures (Table 1). For immunofluorescence results from the same Rab3A^-/- cultures, no trend towards higher values was apparent for GluA2 receptor cluster characteristics (Table 1). However, given the variability of the GluA2 response in cultures from Rab3A^+/+ mice, it would require a much higher number of Rab3A^+/+ and Rab3A^-/- cultures to make any firm conclusions about the role of Rab3A in the homeostatic response of GluA2 receptors.

Having used VGLUT1 immunofluorescence to mark synaptic sites, we could also ask whether there was a presynaptic effect of activity blockade on the size, intensity or integral of the VGLUT1 signal at sites apposed to the previously examined GluA2 postsynaptic sites. In the pooled data from cultures prepared from Rab3A^+/+ mice, we found no change in the size of the VGLUT1-positive regions, and trends towards reduced intensity and integral (Table 2). These data do not support the idea that activity blockade caused an increase in the amount of VGLUT1 per vesicle, although we cannot rule out that a simultaneous reduction in the number of vesicles obscured the effect, since we did not have an independent label for synaptic vesicles. In cultures prepared from Rab3A^-/- mice, the size, intensity and integral of the VGLUT1- positive regions did not significantly change (Table 2).

Table 2.

We have demonstrated here for the first time that the synaptic vesicle protein Rab3A influences the homeostatic regulation of quantal amplitude. What are possible ways that Rab3A could exert its effect? It has previously been shown that exogenous addition of TNFα to hippocampal cultures causes an increase in surface expression of GluA1 receptors (although not GluA2 receptors), and that the homeostatic increase in mEPSC amplitudes is abolished in cultures prepared from the TNFα deletion mouse (Stellwagen et al., 2005; Stellwagen and Malenka, 2006). Furthermore, neurons from TNFα^+/+ mice plated on glial feeder layers derived from TNFα^-/- mice fail to show the increase in mEPSC amplitude after TTX treatment, indicating that the TNFα inducing the receptor increases following activity blockade comes from the glial cells (Stellwagen and Malenka, 2006). It has recently been shown that the source of TNFα is astrocytes rather than microglia (Heir et al., 2024). Rab3A has been detected in astrocytes (Maienschein et al., 1999; Hong et al., 2016), so to determine whether Rab3A is acting via regulating TNFα release from astrocytes, we performed experiments similar to those of Stellwagen and Malenka (2006) (schema illustrated in Figure 6, Left). Under our culturing conditions, the glial feeder layers would be expected to be composed mainly of astrocytes (Heir et al., 2024). We compared the effect of activity blockade on mEPSC amplitudes recorded from cortical neurons from Rab3A^+/+ mice plated on Rab3A^+/+ glial feeder layers (Figure 6A); neurons from Rab3A^+/+ mice plated on Rab3A^-/- glia (Figure 6B), and neurons from Rab3A^-/- mice plated on Rab3A^+/+ glia (Figure 6C). If Rab3A is required for TNFα release from glia, then Rab3A^+/+ neurons plated on Rab3A^-/- glia should not show a homeostatic increase in mEPSC amplitude after treatment with TTX, and any cultures with Rab3A^+/+ glia should have a normal homeostatic response. We found the opposite result: mEPSC amplitudes increased dramatically in cultures where Rab3A was present in neurons (WT on WT, CON, 13.3 ± 0.5 pA, TTX, 16.7 ± 1.2 pA; WT on KO, CON, 13.3 ± 1.0 pA, TTX, 18.8 ± 1.4 pA), but the increase was greatly diminished in cultures where Rab3A was present only in glia (KO on WT, CON, 15.2 ± 1.1 pA, TTX, 16.9 ± 0.7 pA). In the glial feeder layer culture, the loss of Rab3A in the neurons may have caused an increase in mEPSC amplitude at baseline, similar to the Earlybird mutant in dissociated cultures, but this difference was not significant (CON, WT on WT, 13.3 ± 0.5 pA; CON, KO on WT, 15.2 ± 1.1 pA, p = 0.23, Kruskal-Wallis test), so it is also possible this can be attributed to culture to culture variability. Regarding mEPSC frequency, in cultures with Rab3A^+/+ neurons, frequency trended upward after activity blockade, whereas in the culture with Rab3A^-/- neurons, frequency was already increased (although not significantly, CON, WT on WT, 2.54 ± 0.57 sec^-1; CON, KO on WT, 4.46 ± 1.21 sec^-1, p = 0.23, Kruskal-Wallis test), and trended downward after activity blockade.

Figure 6.

In summary, in the absence of Rab3A from glia, the homeostatic plasticity of mEPSC amplitude following activity blockade was normal, whereas in the absence of Rab3A in neurons, homeostatic plasticity was greatly diminished. This result makes it highly unlikely that Rab3A is required for the release of TNFα, or another factor from glia, that induces a homeostatic upregulation of postsynaptic receptors and thereby increases mEPSC amplitude following TTX treatment. Neuronal Rab3A appears to mediate the homeostatic increase in mEPSC amplitude following activity blockade.

Discussion

We found that homeostatic synaptic plasticity of mEPSC amplitude in dissociated mixed cultures of mouse cortical neurons and glia behaved remarkably similar to the mouse NMJ in response to loss of Rab3A function: in cultures from Rab3A^-/- mice, the increase in mEPSC amplitudes following prolonged network silencing by TTX was strongly diminished, and in cultures from Rab3A^Ebd/Ebd mice, basal mEPSC amplitude was increased compared to that of wild-type cultures and was not further modified by TTX treatment. These results suggest that normal function of the synaptic vesicle protein Rab3A is required for the homeostatic increase of mEPSC amplitude in cortical cultures. At the NMJ, the effect of Rab3A on homeostatic upregulation of mEPC amplitude was not mediated by upregulation of postsynaptic receptors. In neurons, the increase in mEPSC amplitude following block of activity was not due to increases in Ca²⁺-permeable GluA1 homomers because acute NASPM application was unable to reverse the TTX-induced increase in amplitude. Imaging of immunofluorescence of GluA2 receptors revealed the response was more variable than that of mEPSC amplitude. The simplest explanation for our findings at both the NMJ and in neurons is that Rab3A regulates a presynaptic contributor to alteration of quantal amplitude during homeostatic synaptic plasticity.

Is the lack of homeostatic plasticity in the Earlybird mutant due to occlusion?

We found that mEPSC amplitude was increased in untreated cultures prepared from Rab3A^Ebd/Ebd mice, compared to mEPSC amplitude in cultures from wild type mice. If there is a limit to how large an mEPSC can become, it is possible that the mutant Rab3A does not affect homeostatic plasticity, only occludes it. Similar increases in mEPSC amplitude at baseline, combined with a failure to increase further after activity blockade, were noted in cultures prepared from mECP2, AKAP, Homer1a, and Arc/Arg3.1 deletion mice (Shepherd et al., 2006; Hu et al., 2010; Xu and Pozzo-Miller, 2017; Sanderson et al., 2018) as well as in cultures treated with ubiquitin proteasome inhibitors (Jakawich et al., 2010) or microRNA 186-5p (Silva et al., 2019), suggesting the possibility of a generalized phenomenon in which increased mEPSC amplitude is induced when homeostatic regulation is disrupted. Since we did not observe such an increase in mEPSC amplitude at baseline in cultures from Rab3A^-/- mice, it remains a possibility that the point mutant may bind to novel partners, causing activities that would not be either facilitated or inhibited by Rab3A. Still, it is a strong coincidence that the novel activity of the mutant Earlybird Rab3A would affect mEPSC amplitude, the same characteristic that is modulated by activity blockade in a Rab3A dependent manner. Although we cannot rule out occlusion, an alternate interpretation is that the presence of the mutant protein mimics the condition of activity blockade.

Homeostatic plasticity of postsynaptic receptors was less consistent than that of mEPSC amplitude

While we found that the effect of TTX treatment on mEPSC amplitude and GluA2 receptor levels were of similar magnitude, the effect on GluA2 receptor levels was not statistically significant for the pooled data sets acquired in the same cultures, nor in a larger data set containing 5 cultures. In light of the extensive literature documenting increases in GluA1 or GluA2 receptors, or both, after activity blockade, the less than convincing increase in GluA2 receptor levels in the current work came as a surprise. The reason for the lack of significance was that the increase in receptors was more variable. We are aware of three possible explanations for greater variability. 1) The number of synaptic sites sampled was smaller for the imaging data set. This was because we limited our analysis to synaptic receptors on the primary dendrite, where the increase in sensitivity to exogenously applied glutamate was shown to be greatest (see Figure 3 in (Turrigiano et al., 1998)). To address this issue, we limited our sample size of mEPSCs in the matched experiments to the same number per cell as the mean sample size per cell for imaging, but there was still a discrepancy in the effect of TTX on mEPSCs vs. receptors. 2) The small sample of only 3 cultures was not enough to reveal the increase in receptors. We attempted to address this by adding 2 more cultures. Although both additional cultures showed increases in GluA2 receptor cluster size, the magnitudes of the increases were not large, and when cells from these cultures were included in the pooled data set, the p value was not substantially improved. The only remaining possibility is: 3) The complexity involved in processing cultures for immunofluorescence introduces variability that obscures the true effect. One can get around technical issues related to immunofluorescence processing by performing Western blots, but then any changes detected could be due to upregulation of extrasynaptic receptors. What remains unclear is why our data were impacted by this but data of other studies were not. Some other studies have used the number of synaptic sites or puncta as the sample size (for example, (Hou et al., 2008; Ibata et al., 2008; Wang et al., 2019)). When we analyzed our data after including as few as 6 randomly selected cluster size measurements per cell, a Kruskal-Wallis test gave highly significant p values for 3 cultures (p = 0.001, n = 174 sites (6 *29 cells)) and 5 cultures (p = 0.005, n = 294 sites (6 *49 cells)). We found only a single study that had comparable sample sizes for receptors and mEPSC amplitudes, and the p value was < 0.01 for mEPSC amplitudes (n = 9 and 10 cells for CON and TTX, respectively), but five times higher (< 0.05) for receptor data (n = 13 and 14 cells for CON and TTX) (Xu and Pozzo-Miller, 2017). This single example is consistent with our suggestion that measurements of changes in receptors after activity blockade are more variable than measurements of mEPSC amplitudes. In sum, our data suggest that TTX treatment of mouse cortical cultures consistently leads to an increase in mEPSC amplitudes, and sometimes leads to an increase in synaptic GluA2 receptor cluster size. Increased receptors likely contribute to increases in mEPSC amplitudes, but our current data, coupled with our previous findings at the mouse neuromuscular junction, support the idea that there are additional sources contributing to the homeostatic increase in quantal size.

Neuronal, not glial, Rab3A is required for homeostatic regulation of quantal amplitude

Astrocytic release of TNFα was shown to mediate the increase in mEPSC amplitudes in prolonged TTX-treated cultures of dissociated hippocampal neurons and cultures of hippocampal slices (Stellwagen and Malenka, 2006; Heir et al., 2024), but we found that loss of Rab3A in glia did not disrupt the increase in mEPSC amplitude after activity blockade. Further, we previously showed that the homeostatic increase in NMJ mEPC amplitude was completely normal in the absence of TNFα (Wang et al., 2011). This leaves neuronal Rab3A as the potential controller of homeostatic regulation of quantal amplitude.

Is Rab3A acting in the presynaptic cell to cause homeostatic increase in mEPSC amplitude?

Our current results support the surprising possibility that the presynaptic vesicle protein Rab3A either regulates postsynaptic AMPARs or regulates a presynaptic contributor to homeostatic synaptic plasticity. To our knowledge, there is no evidence localizing Rab3A to postsynaptic dendritic sites. Other presynaptic molecules, such as SNARE proteins, synaptotagmins, and NSF, have been identified in the postsynaptic compartment (Lledo et al., 1998; Nishimune et al., 1998; Osten et al., 1998; Song et al., 1998; Araki et al., 2010; Kennedy et al., 2010; Suh et al., 2010; Jurado et al., 2013; Hussain and Davanger, 2015; Gu et al., 2016; Wu et al., 2017). A recent report implicates Rab3A in the localization of plasma membrane proteins, including the EGF receptor, in rafts in HEK cells and Jurkat-T cells, which indicates Rab3A can be found in other specialized locations besides the presynaptic nerve terminal (Diaz-Rohrer et al., 2023). Without additional experiments targeting Rab3A deletion to the pre- or post-synaptic compartment, we cannot rule out that Rab3A is regulating homeostatic plasticity in mouse cortical cultures by acting postsynaptically on receptor trafficking. However, given our previous finding of a lack of any increase in AChR after TTX treatment the NMJ (Wang et al., 2011), the simplest interpretation is that in these two situations, Rab3A is required for an increase in the presynaptic quantum rather than an increase in postsynaptic receptors.

The presynaptic quantum, or the amount of transmitter released during a single fusion event, is an important contributor to quantal size. The amount of transmitter released during vesicle fusion can be affected by the kinetics of the fusion pore opening (Chang et al., 2017). We previously used amperometric measurements in mouse adrenal chromaffin cells to show loss of Rab3A increased the occurrence of very small amplitude fusion pore feet (Wang et al., 2008). Paired with our finding that there is an increase in the occurrence of very slow-rising, abnormally shaped mEPCs at the neuromuscular junction of Rab3A^-/- mice, our data suggest that small synaptic vesicles may have a fusion pore step under certain circumstances (see also (Chiang et al., 2018)). If activity blockade causes an increase in mEPSC amplitude due to a more rapid opening of a fusion pore, or a larger fusion pore conductance, it is possible that Rab3A is required for this modulation.

Another way to increase the presynaptic quantum is to increase levels of the transmitter transporter (Daniels et al., 2004; De Gois et al., 2005; Wilson et al., 2005; Hartman et al., 2006). However, we found that immunofluorescence for the glutamate transporter, VGLUT1, was not increased after activity blockade under our experimental conditions. A similar lack of increase in VGLUT1 levels, relative to synapsin, was previously observed in hippocampal cultures treated with NBQX, an AMPA receptor blocker (see supplemental data, (Wilson et al., 2005)). Taken together with our findings, it appears that there is not strong evidence that the amount of VGLUT1 at synapses is a contributing factor to the increase in mEPSC amplitude in neuronal cultures after activity blockade with TTX.

Finally, the amount of transmitter released by a vesicle could be increased due to larger vesicle size. Larger vesicle diameter has been observed following activity disruption in multiple paradigms, including ocular deprivation, cortical ablation, and immobilization (Kawana et al., 1971; Lloret and Saavedra, 1975; Cheresharov et al., 1978). Loss of Rab3 family members is associated with increased vesicle size (Riedel et al., 2002; Schonn et al., 2010). Finally, an increase in volume has been shown to increase miniature endplate junctional current amplitude at the Drosophila NMJ (Karunanithi et al., 2002). Rab3A could be required for an activity-dependent increase in vesicle size, but the increase in diameter needed for a 25% increase in transmitter, and therefore mEPSC amplitude, would be difficult to detect.

The number of molecules shown to be required for homeostatic synaptic plasticity of quantal size in neuronal cultures may have surpassed ∼20 (see (Koesters et al., 2024) for a recent summary), but fall into two main categories: molecules involved in glutamate receptor expression or trafficking, and signaling molecules. To our knowledge, this is the first evidence that a presynaptic protein is participating in this process. Homeostatic synaptic plasticity is becoming increasingly implicated in essential normal functions such as sleep (Tononi and Cirelli, 2014; Diering et al., 2017; Torrado Pacheco et al., 2021), and pathological conditions such as epilepsy, neuropsychiatric disorders, Huntington’s, and alcohol use disorder (Trasande and Ramirez, 2007; Fernandes and Carvalho, 2016; Wang et al., 2017; Lovinger and Abrahao, 2018; Smith-Dijak et al., 2019; Lignani et al., 2020; Suzuki et al., 2021; Kavalali and Monteggia, 2023). Therefore, our identification of a novel Rab3A-dependent regulatory pathway in homeostatic synaptic plasticity may have important therapeutic implications. Our findings also further cement the importance of Rab3A in activity-dependent modulation of synaptic strength.

Materials and methods

Animals

To determine the role of Rab3A in homeostatic plasticity in mouse cortical cultures, we employed two distinct genetic mouse strains with altered Rab3A function. Rab3A deletion mice were generated as follows: Rab3A^+/- heterozygous mice were bred and genotyped as previously described (Kapfhamer et al., 2002; Wang et al., 2008), and maintained as a colony using multiple heterozygous breeding pairs. Homozygous Rab3A^+/+ or Rab3A^-/- mouse pups were obtained by breeding Rab3A^+/+ pairs and Rab3A^-/- pairs, respectively, so that the pups were the first-generation progeny of a homozygous mating. This protocol minimizes the tendency of multiple generations of Rab3A^+/+ and Rab3A^-/- homozygous breedings to produce progeny that are more and more genetically distinct. Rab3A^Ebd/Ebdmice were identified in an EU-mutagenesis screen of C57BL/6J mice, and after a cross to C3H/HeJ, were backcrossed for 3 generations to C57BL/6J (Kapfhamer et al., 2002). Rab3A^+/Ebd heterozygous mice were bred at Wright State University and genotyped in a two-step procedure: 1. a PCR reaction (forward primer: TGA CTC CTT CAC TCC AGC CT; reverse primer: TGC ACT GCA TTA AAT GAC TCC T) followed by 2. a restriction digest with enzyme Bsp1286I (New England Biolabs) that distinguishes the Earlybird mutant by its different base-pair products. Rab3A^+/- mice were backcrossed with Rab3A^+/+ mice from the Earlybird heterozygous colony for 11 generations in an attempt to establish a single wild type strain, but differences in mEPSC amplitude and adrenal chromaffin cell calcium currents persisted, likely due to genes that are close to the Rab3A site, resulting in two wild type strains: 1. Rab3A^+/+ from the Rab3A^+/- colony, and 2. Rab3A^+/+ from the Rab3A^+/Ebd colony.

Primary Culture of Mouse Cortical Neurons

Primary dissociated cultures of mixed neuronal and glia populations were prepared as previously described (Hanes et al., 2020). Briefly, postnatal day 0-2 (P0-P2) Rab3A^+/+, Rab3A^-/- or Rab3A^Ebd/Ebd neonates were euthanized by rapid decapitation, as approved by the Wright State University Institutional Animal Care and Use Committee, and brains were quickly removed. Each culture was prepared from the cortices harvested from two animals; neonates were not sexed. Cortices were collected in chilled Neurobasal-A media (Gibco) with osmolarity adjusted to 270 mOsm and supplemented with 40 U/ml DNAse I (ThermoFisher Scientific). The tissues were digested with papain (Worthington Biochemical) at 20 U/ml at 37°C for 20 minutes followed by trituration with a sterile, fire-polished Pasteur pipette, then filtered through a 100 μm cell strainer, and centrifuged at 1100 rpm for 2 minutes. After discarding the supernatant, the pellet was resuspended in room temperature Neurobasal-A media (270 mOsm), supplemented with 5% fetal bovine serum for glia growth, and 2% B-27 supplement to promote neuronal growth (Gibco), L-glutamine, and gentamicin (ThermoFisher Scientific). Neurons were counted and plated at 0.15 * 10⁶ cells/coverslip onto 12 mm coverslips pre-coated with poly-L-lysine (BioCoat, Corning). This plating density produces a “high density” culture characterized by a complex mesh of neuronal processes criss-crossing the field of view, completely filling the space between cell bodies (see Figure 4). We found in preliminary experiments that an increase in mEPSC amplitude in mouse cortical cultures was inconsistent when cell density was low, likely due to lower levels of baseline activity, although it is a limitation of this study that we did not directly measure activity levels in cultures prepared from Rab3A^+/+, Rab3A^-/- or Rab3A^Ebd/Ebd mice, and therefore do not know if differences in results are due to differences in activity levels. While unlikely for Rab3A^+/+ and Rab3A^-/- studies, given the mEPSC amplitude in untreated cultures was relatively small, it remains possible that in Rab3A^Ebd/Ebd studies, the larger mEPSC amplitude in untreated cultures was due to a loss of activity in these cultures. The culture media for the first day (0 DIV) was the same as the above Neurobasal-A media supplemented with FBS, B-27, L-glutamine, and gentamicin, and was switched after 24 hours (1 DIV) to media consisting of Neurobasal-A (270 mOsm), 2% B-27, and L-glutamine without FBS to avoid its toxic effects on neuronal viability and health (Stellwagen and Malenka, 2006). Half of the media was changed twice weekly and experiments were performed at 13-14 DIV. Two days prior to experiments, tetrodotoxin (TTX) (500 nM; Tocris), a potent Na⁺ channel blocker, was added to some cultures to chronically silence all network activity and induce homeostatic synaptic plasticity mechanisms, while untreated sister cultures served as controls. Cultures prepared from mutant mice were compared with cultures from wild-type mice from their respective colonies. Note that the cultures comprising the Rab3A^+/+ data here are a subset of the data previously published in Hanes et al., 2020. This smaller data set was restricted to the time period over which cultures were also prepared from Rab3A^-/- mice.

Preparation of Glial Feeder Layers

Glial feeder layers were prepared from the cortices of P0-P2 Rab3A^+/+ or Rab3A^-/- mouse pups as described previously (Stellwagen and Malenka, 2006). Briefly, cortices were dissected and cells were dissociated as described above. Cell suspensions of mixed neuronal and glial populations were plated onto glass coverslips pre-coated with poly-L-lysine in Dulbecco’s Modified Eagle Media (ThermoFisher Scientific) supplemented with 5% FBS (to promote glial proliferation and to kill neurons), L-glutamine, and gentamicin, and maintained in an incubator at 37°C, 5% CO₂; cultures were maintained in this manner for up to 1 month to generate purely glial cultures (all neurons typically died off by 7 DIV). Culture media was replaced after 24 hours, and subsequent media changes were made twice weekly, replacing half of the culture media with fresh media. Feeder layers were not used for neuronal seeding until all native neurons were gone and glia approached 100% confluency (visually inspected).

Plating of Neurons on Glial Feeder Layers

Cortical neurons were obtained as described above. The cell pellet obtained was resuspended in Neurobasal-A (osmolarity adjusted to 270 mOsm) containing B27 (2%, to promote neuronal growth), L-glutamine, and 5-fluorodeoxyuridine (FdU, a mitotic inhibitor; Sigma). Addition of FdU was used to prevent glial proliferation and contamination of the feeder layer with new glia, promoting only neuronal growth on the feeder layers (FdU-containing media was used for the maintenance of these cultures and all subsequent media changes). Glial culture media was removed from the feeder layer cultures, and the neuronal cell suspension was plated onto the glial feeder cultures. The culture strategy used to distinguish the relative roles of neuronal and glial Rab3A is outlined in Figure 8, Left. At 1 DIV, all of the culture media was removed and replaced with fresh Neurobasal-A media containing FdU described above, and half of the media was replaced twice per week for all subsequent media changes. Cultures were maintained in a 37°C, 5% CO₂ incubator for 13-14 DIV.

Whole-Cell Voltage Clamp to Record mEPSCs

At 13-14 DIV, mEPSCs from TTX-treated and untreated sister cultures of Rab3A^+/+ or Rab3A^-/- neurons from the Rab3A^+/- colony, or Rab3A^+/+ or Rab3A^Ebd/Ebd neurons from the Rab3A^+/Ebd colony, were recorded via whole-cell voltage clamp to assess the role of Rab3A in homeostatic synaptic plasticity. Recordings were taken from pyramidal neurons, which were identified visually by a prominent apical dendrite; images were taken of all cells recorded from. Cells were continuously perfused with a solution consisting of (in mM): NaCl (115), KCl (5), CaCl₂ (2.5), MgCl₂ (1.3), dextrose (23), sucrose (26), HEPES (4.2), pH = 7.2 (Stellwagen and Malenka, 2006). On the day of recording, the osmolarity of the media from the cultures was measured (normally 285 – 295 mOsm) and the perfusate osmolarity was adjusted to match the culture osmolarity to protect against osmotic shock to the neurons. To isolate glutamatergic mEPSCs, TTX (500 nM) and picrotoxin (50 μM), were included in the perfusion solution to block action potentials and GABAergic currents, respectively. The NMDA receptor antagonist, APV, was not included in the perfusion solution because in pilot studies, all mEPSCs were blocked by 10 µM CNQX and 50 μm picrotoxin, demonstrating no APV-sensitive mEPSCs were present (data not shown). Previous studies have described NMDA mEPSCs in neuronal cultures, but recordings were performed in 0 Mg²⁺ (Watt et al., 2000; Sutton et al., 2006). In our recordings, the presence of extracellular Mg²⁺ (1.3 mM) and TTX would mean that the majority of NMDA receptors are blocked by extracellular Mg²⁺ at resting Vm. Patch electrodes (3.5 – 5 MΩ) were filled with an internal solution containing (in mM): K-gluconate (130), NaCl (10), EGTA (1), CaCl₂ (0.132), MgCl₂ (2), HEPES (10), pH = 7.2. Osmolarity was adjusted to 10 mOsm less than the perfusion solution osmolarity, which we have found improves success rate of achieving low access resistance recordings. Neurons were clamped at a voltage of −60 mV using an Axopatch 200B patch-clamp (Axon Instruments), recorded from for 2 – 5 minutes, and data were collected with Clampex 10.0/10.2 (Axon Instruments).

NASPM application

The antagonist of Ca²⁺-permeable AMPA receptors, N-naphthyl acetylspermine (NASPM, 20 μM; Tocris), was applied during recordings in a subset of experiments. NASPM is a synthetic analog of Joro Spider Toxin (JSTX) (Koike et al., 1997), and chemicals in this family block Ca²⁺ permeable glutamate receptors (Blaschke et al., 1993; Herlitze et al., 1993; Iino et al., 1996). The presence of the edited GluA2 subunit disrupts both the Ca²⁺ permeability and the sensitivity to JSTX and related substances (Blaschke et al., 1993; Bochet et al., 1994; Jonas and Burnashev, 1995). Therefore, 20 μM NASPM is expected to completely block AMPA receptors containing GluA1, GluA3 or GluA4 subunits (Hollmann et al., 1991; Burnashev et al., 1992), or the Kainate receptors containing GluA5 or GluA6 (Koike et al., 1997; Sun et al., 2009), but be ineffective against any heteromer or homomer containing GluA2 subunits. Because the effect of the spider toxins and their analogues are use-dependent (Herlitze et al., 1993; Iino et al., 1996; Koike et al., 1997), NASPM was applied with a depolarizing high K⁺ solution (25 mM KCl, 95 mM NaCl) which we expected to trigger release of glutamate and opening of glutamate-activated receptors, allowing entry of NASPM into the pore. Baseline recordings were performed for 2 minutes in our standard perfusate, followed by perfusion of NASPM + High K⁺ solution for 45 seconds, and then perfusion of a NASPM only solution for 5 minutes, after which recording was recommenced for 5 minutes (because we found in pilot experiments that frequency was reduced following NASPM application).

Analysis of mEPSCs

Miniature excitatory postsynaptic currents (mEPSCs) were manually selected using Mini Analysis software (Synaptosoft), a standard program used for mEPSC event detection and analysis. Records were filtered at 2 kHz using a low-pass Butterworth filter prior to selection. The program detection threshold was set at 3 pA but the smallest mEPSC selected was 4.01 pA. A fully manual detection process means that the same criterion (“does this look like an mEPSC?”) is applied to all events, not just the false positives or the false negatives, which prevents the bias from being primarily at one end or the other of the range of mEPSC amplitudes. It is important to note that when performing the MiniAnalysis process, the researcher did not know whether a record was from an untreated cell or a TTX-treated cell.

Immunofluorescence, microscopy, and data analysis

Primary cultures of mouse cortical neurons were grown for 13-14 DIV. Antibodies to GluA2 (mouse ab against N-terminal, EMD Millipore) were added directly to live cultures at 1:40 dilution, and incubated at 37 °C in a CO₂ incubator for 45 minutes. Cultures were rinsed 3 times with PBS/5% donkey serum before being fixed with 4% paraformaldehyde. After 3 rinses in PBS/5% donkey serum, cultures were incubated in CY3-labeled donkey-anti-mouse secondary antibodies for 1 hour at room temperature, rinsed in PBS/5% donkey serum, permeabilized with 0.2% saponin, and incubated in chick anti-MAP2 (1:2500, AbCAM) and rabbit anti-VGLUT1 (1:4000, Synaptic Systems) for 1 hour at room temperature in PBS/5% donkey serum. After rinsing with PBS/5% donkey serum, coverslips were incubated with 488-anti chick and CY5-anti rabbit secondary antibodies for 1 hour at room temperature, rinsed, blotted to remove excess liquid, and inverted on a drop of Vectashield (Vector Labs). Coverslips were sealed with nail polish and stored at 4 °C for < 1 week before imaging. All secondary antibodies were from Jackson Immunoresearch and were used at 1:225 dilution.

Coverslips were viewed on a Fluoview FV1000 laser scanning confocal microscope with a 60x oil immersion, 1.35 NA objective. Once a pyramidal neuron was identified and a single confocal section of the cell acquired (see Top, Figure 4), Fluoview 2.1 software was used to zoom in on the primary dendrite (5X) and a series of confocal sections were taken every 0.5 μm. Images were analyzed offline with ImagePro 6 (Cybernetics). The composite image was used to locate synaptic sites containing both VGLUT1 and GluA2 immunoreactivity in close apposition to each other and to the primary dendrite or a secondary branch identified with MAP2 immunoreactivity. An area of interest (AOI) was manually drawn around the GluA2 cluster in the confocal section in which it was the brightest. The AOIs for a dendrite were saved in a single file; the AOI number and the confocal section it was associated with were noted for later retrieval. For quantification, AOIs were loaded, an individual AOI was called up on the appropriate section, and the count/measurement tool used to apply a threshold. Multiple thresholds were used, 400-550, but we found that within a given experiment, the magnitude of the TTX effect was not altered by different thresholds, therefore, all data presented here use a threshold of 450. Pixels within the cluster that were above the threshold were automatically outlined, and size, average intensity, and integral of the outlined region saved to Origin2020 (OriginLab) file for calculating mean values. For analyzing VGLUT1 sites, previous AOIs were examined, and if not directly over the VGLUT1 site, moved to encompass it (this happened when GluA2 and VGLUT1 were side by side rather than overlapping). The threshold was 200 for all VGLUT1 data except Rab3A^+/+ Culture #1, where threshold was 400. There was no threshold that worked for both that culture and the other cultures, with too much of the background regions being included for Culture #1 with a threshold of 200, and too little of the synaptic site area being included for the other Cultures with a threshold of 400.

Statistics

In our previous publication (Hanes et al., 2020), we described the characteristics of homeostatic plasticity of mEPSC amplitudes in mouse cortical cultures using multiple mEPSC amplitude quantiles from each cell to create data sets with thousands of samples. We compared the cumulative distribution functions for mEPSC amplitudes in untreated vs. TTX- treated cultures, as well as sorting the values from smallest to largest, and plotted TTX values vs. control values in the rank order plot first described in (Turrigiano et al., 1998) to demonstrate synaptic scaling. Finally, we plotted the ratio of TTX values/control values vs. control values to show that the ratio, which represents the effect of TTX, increased with increasing control values, which we termed “divergent scaling.” Here, we wanted to avoid the inflation of sample size caused by pooling multiple mEPSCs per cell in order to statistically compare the effects of TTX in cultures of wild type mice, Rab3A^-/- mice and Rab3A^Ebd/Ebd mutant mice. Therefore, we computed the mean mEPSC amplitude for each cell and compared means from untreated and TTX-treated cultures with the non-parametric Kruskal-Wallis test, with N = the number of cells. The overall means are presented ± SEM in the text, and p values are displayed above each plot. The same process was carried out for the characteristics of synaptic GluA2 receptor clusters and VGLUT1 sites in imaging experiments, with N = the number of dendrites (one dendrite was sampled for each cell). Although we quantified three characteristics of the GluA2 clusters and VGLUT1 sites, size, average intensity, and integral, we have not corrected p values for multiple comparisons. Means are presented as box plots + data, with the box at 25 and 75 percentiles, whiskers at 10 and 90 percentiles, a dot indicating the mean, and a line indicating the median. In Figure 3, mEPSC amplitudes were measured within the same cell before and after NASPM application, so a paired t test was used to compare the pre- and post-NASPM mEPSC amplitudes. In Figure 5A-C the mean mEPSC amplitude for untreated and TTX-treated coverslips in the same culture are compared with a paired t test. Statistics were computed and plots were created using Origin2020 (OriginLab).

Acknowledgements

The authors thank E. Whitlock and H. Ghouse for their contributions to confocal image analyses. This work was partially supported by NINDS P01 NS057228, Tim Cope, PI, KE, PI project 4.

Additional information

Author contributions

A.G.K., K.L.E. and M.M.R. designed research; A.G.K. performed research; A.G.K. and K.L.E. analyzed data; A.G.K, K.L.E., and M.M.R. wrote the paper.