A trans-synaptic nanocolumn aligns neurotransmitter release to receptors

The average estimated diameter of RIM1/2 nanoclusters is very close to the reported size of PSD-95 and AMPA receptor nanoclusters.

“The average estimated diameter of ~80 nm for RIM1/2 NCs was very close to the reported size of PSD-95 and AMPA receptor (AMPAR) NCs4–6.”

• cites [4] MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013) sourcePartly supported

  The Results section, “Scaffold molecules are organized in subsynaptic clusters” (page unavailable), reports a PSD-95 subcluster diameter of 83 ± 4 nm, close to the citing sentence’s ~80 nm. The Results section, “Subsynaptic scaffolding domains concentrate synaptic AMPARs” (page unavailable), reports AMPAR enrichment within those domains but does not give a separate AMPAR nanocluster diameter. This source therefore supports the PSD-95 size comparison, but not the stated AMPAR nanocluster size. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [5] Nair, D. et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J. Neurosci. 33, 13204–13224 (2013) sourcePartly supported

  The ‘Subsynaptic AMPAR clusters’ section reports AMPAR nanodomain axes near 80 nm, supporting that comparison. But ‘Nanoscale co-organization of PSD95 and AMPARs’ (Fig. 7E) reports PSD95 subdomain axes of 158.2 and 102.2 nm and explicitly describes the AMPAR nanodomains as smaller. The copy does not support treating both reported sizes as very close to 80 nm. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [6] Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013) sourceDisputed

  The Results section (page unavailable) reports a mean diameter of about 200 nm for palmitoylated PSD-95 nanodomains, not a size very close to the citing sentence’s ~80 nm. It reports that AMPAR clusters associate with those nanodomains but gives no AMPAR-cluster diameter. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Presynaptic RIM nanoclusters closely align with concentrated postsynaptic receptors and scaffolding proteins.

“These presynaptic RIM nanoclusters closely aligned with concentrated postsynaptic receptors and scaffolding proteins4–6, suggesting a transsynaptic molecular “nanocolumn.””

• cites [4] MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013) sourcePartly supported

  The Results section reports that AMPA receptors are enriched in postsynaptic PSD-95 scaffold clusters. The Discussion proposes pre–post alignment as a possibility but also says these clusters may concentrate receptors toward the synapse’s center rather than specifically align them with release sites. It does not establish close alignment with presynaptic RIM nanoclusters. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [5] Nair, D. et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J. Neurosci. 33, 13204–13224 (2013) sourcePartly supported

  The Abstract reports concentrated postsynaptic AMPA-receptor nanodomains that are often, but not always, colocalized with PSD95 scaffold clusters. The Discussion only proposes a possible correlation with presynaptic release sites; it does not establish that presynaptic RIM nanoclusters closely align with those postsynaptic clusters. No page numbers are available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [6] Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013) sourcePartly supported

  The Results section on subsynaptic nanodomains (Fig. 4F–H; page unavailable) shows postsynaptic PSD-95 nanodomains associated with AMPA receptors and apposed to presynaptic vGlut1 labeling. It does not identify presynaptic RIM nanoclusters or show that those clusters closely align with the postsynaptic proteins. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

PSD-95 nanoclusters concentrate higher density of receptors.

“as the PSD-95 NCs concentrate higher density of receptors7.”

• cites [7] Südhof, T. C. The presynaptic active zone. Neuron 75, 11–25 (2012) sourceCould not verify

  The passages from “Membrane Proteins of Active Zones” discuss receptors concentrated in presynaptic active zones, not receptors concentrated by PSD-95 nanoclusters. Searches for “PSD-95 nanoclusters” and “PSD-95 postsynaptic density receptors clustering” found no passage in our copy addressing the claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

The alignment of pre- and postsynaptic nanoscale subdomains suggests that even small synapses may be composed of dynamic functional modules.

“The alignment of pre and postsynaptic nanoscale subdomains4–6 suggests that even small synapses may be composed of dynamic functional modules28,29.”

• cites [4] MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013) sourceSupported
• cites [5] Nair, D. et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J. Neurosci. 33, 13204–13224 (2013) sourcePartly supported

  The Discussion (page unavailable) describes dynamic AMPAR nanodomains that may act as functional units activated by single-vesicle release, supporting the proposed module concept. The cited paper does not specifically establish that this organization occurs in small synapses; the sentence’s alignment claim is attached to other citations. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [6] Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013) sourceSupported
• cites [28] Tarr, T. B., Dittrich, M. & Meriney, S. D. Are unreliable release mechanisms conserved from NMJ to CNS? Trends Neurosci. 36, 14–22 (2013) sourceSupported
• cites [29] Lisman, J. & Raghavachari, S. A unified model of the presynaptic and postsynaptic changes during LTP at CA1 synapses. Sci. STKE 2006, re11 (2006) sourceNot checked — no full text of the source

Sensitivity of PSD-95 nanocluster size to latrunculin suggests that the spine cytoskeleton is engaged at the base of the column.

“Nevertheless, sensitivity of PSD-95 NC size to latrunculin4 further suggests that the spine cytoskeleton is engaged at the “base” of the column.”

• cites [4] MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013) sourceSupported

The impact of presynaptic nanoscale organization and confined vesicle sites on the synapse as a whole depends strongly on whether RIM nanoclusters align with postsynaptic receptor nanoclusters.

“For the synapse as a whole, the impact of presynaptic nanoscale organization and confined vesicle sites (Fig. 1, 2) will depend strongly on whether these RIM NCs align with postsynaptic receptor NCs4.”

• cites [4] MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013) sourcePartly supported

  The Results section (‘Clustered distribution shapes postsynaptic responses’; page not provided) reports simulated currents nearly twice as large when vesicles released glutamate over AMPA-receptor clusters rather than off-cluster. The Discussion identifies pre–post alignment as a way to maximize transmission, but does not establish that this effect depends on alignment of RIM nanoclusters specifically; it also reports an advantage for clustered receptors even with randomly distributed release sites. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Spontaneous release in TTX most likely arises from single vesicle fusion.

“we compared them with spontaneous release in TTX (Fig. 2a–c), which most likely arises from single vesicle fusion13.”

• cites [13] Leitz, J. & Kavalali, E. T. Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles . eLife 3, e03658 (2014) sourceSupported

Evoked Pr is significantly positively correlated with Syn1a area.

“evoked Pr was significantly positively correlated with Syn1a area, as reported previously50”

• cites [50] Ermolyuk, Y. S. et al. Differential triggering of spontaneous glutamate release by P/Q-, N- and R-type Ca2+ channels. Nat. Neurosci. 16, 1754–1763 (2013) sourceCould not verify

  The Results section on modeling action potential-evoked release reports vesicular fusion probabilities for different channel-distribution models, but not a correlation with Syn1a area. Searches for “Syn1a,” synapsin area, and evoked release probability versus bouton area found no passage reporting the claimed correlation. Page numbers were unavailable in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

The extent of ga(r) over 1, r0 for ga(r0)=1, is related to the pattern size of the internal heterogeneity.

“The extent of ga(r) over 1, i.e. r0 for ga(r0) =1, is related to the pattern size of the internal heterogeneity (Extended Data Figure 2b–c)39.”

• cites [39] Veatch, S. L. et al. Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One 7, e31457 (2012) sourceSupported

Long-term potentiation can be induced via APV withdrawal and glycine stimulation.

“we induced long-term potentiation via APV withdrawal and glycine stimulation19”

• cites [19] Araki, Y., Zeng, M., Zhang, M. & Huganir, R. L. Rapid dispersion of SynGAP from synaptic spines triggers AMPA receptor insertion and spine enlargement during LTP. Neuron 85, 173–189 (2015) sourcePartly supported

  The Experimental Procedures section (page unavailable) describes inducing LTP by perfusing hippocampal neurons with glycine-containing, magnesium-free solution. The paper lists APV as a reagent and reports that APV blocks chemLTP-induced SynGAP dispersion, but our copy does not describe APV withdrawal as part of the induction method. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The relationship between channel opening probability and distance from a release site is Po(r)=0.42e-r/88nm.

“Central to this approach is the relationship between channel opening probability and its distance from a release site, determined previously by stochastic modeling approaches3,16,47: (6)Po(r)=0.42e-r/88nm”

• cites [3] Franks, K. M., Stevens, C. F. & Sejnowski, T. J. Independent sources of quantal variability at single glutamatergic synapses. J. Neurosci. 23, 3186–3195 (2003) sourceSupported
• cites [16] Raghavachari, S. & Lisman, J. E. Properties of quantal transmission at CA1 synapses. J. Neurophysiol. 92, 2456–2467 (2004) sourceNot checked — no full text of the source
• cites [47] Xie, X., Liaw, J.-S., Baudry, M. & Berger, T. W. Novel expression mechanism for synaptic potentiation: alignment of presynaptic release site and postsynaptic receptor. Proc. Natl Acad. Sci. USA 94, 6983–6988 (1997) sourcePartly supported

  The Results report simulations in which receptor distance from the release site affects AMPA-mediated responses, and the Discussion says response amplitude increases with proximity. But our copy does not give an opening-probability equation or the claimed 0.42 coefficient and 88-nm decay length; its 42% figure is a comparison of EPSC amplitudes, not an opening probability. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The average estimated diameter of RIM1/2 nanoclusters is approximately 80 nm.

“The average estimated diameter of ~80 nm for RIM1/2 NCs was very close to the reported size of PSD-95 and AMPA receptor (AMPAR) NCs4–6.”

Key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometer-scaled subregions of the presynaptic active zone.

“Using localization microscopy, we report here that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometer-scaled subregions of the presynaptic active zone.”

The measured autocorrelation function of the RIM1/2 distribution showed significant nonuniformity compared to random ensembles.

“The measured ACF showed significant nonuniformity compared to random ensembles (Fig. 1d).”

The distribution of RIM1/2 within the active zone, measured as 3D local density, is distinctively nonuniform with notable high-density peaks characterized as nanoclusters.

“The distribution of RIM1/2 within the AZ, measured as 3D local density, was distinctively nonuniform with notable high-density peaks, which we characterized as nanoclusters (NCs, Fig. 1c, e).”

NMDA receptor activation triggers distinct phases of plasticity in which postsynaptic reorganization is followed by transsynaptic nanoscale realignment.

“Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by transsynaptic nanoscale realignment.”

Following the 25-minute recovery, the enrichment index of RIM1/2 with respect to PSD-95 nanoclusters increased with a corresponding increase in the percentage of enriched PSD-95 nanoclusters.

“However, following the 25-minute recovery, the enrichment index of RIM1/2 with respect to PSD-95 NCs increased with a corresponding increase in the percentage of enriched PSD-95 NCs (Fig. 4g–h).”

Following acute NMDA receptor activation, postsynaptic nanostructure was markedly disrupted, with the synaptic cluster volume of PSD-95 and the number, volume, and protein density of PSD-95 nanoclusters all reduced.

“Following this stimulus, postsynaptic nanostructure was markedly disrupted in the generally opposite manner, with the synaptic cluster volume of PSD-95 and the number, volume, and protein density of PSD-95 NCs all reduced (Figs. 4d–f, Suppl. Table 3).”

Randomizing nanocluster positions and out-of-nanocluster molecules, while keeping localizations within nanocluster borders intact, abolished the pair cross-correlation function to chance level.

“Randomizing NC positions and out-of-NC molecules (keeping localizations within NC borders intact) abolished the PCF to chance level, while randomizing just the out-of-NC molecules only modestly reduced the PCF, indicating that the precise positioning of the NCs themselves dominate the overall correlation of protein distributions (Fig. 3c–d).”

These LTP-induced changes were prevented by co-application of the NMDAR antagonist APV.

“These changes were prevented by co-application of the NMDAR antagonist APV (Fig 4a–c, Extended Data Fig. 10m).”

Similar results were found when studying NMDA-induced changes on RIM1/2 and GluA2/3 alignment.

“Similar results were found when we studied NMDA-induced changes on RIM1/2 and GluA2/3 alignment (Extended Data Fig. 10a–h).”

Isolated non-synaptic small groups of localizations showed a weaker autocorrelation function that was significant over a much smaller distance.

“Isolated non-synaptic small groups of localizations showed a weaker ACF that was significant over a much smaller distance (Fig. 1d).”

Randomizing just the out-of-nanocluster molecules only modestly reduced the pair cross-correlation function.

“Randomizing NC positions and out-of-NC molecules (keeping localizations within NC borders intact) abolished the PCF to chance level, while randomizing just the out-of-NC molecules only modestly reduced the PCF, indicating that the precise positioning of the NCs themselves dominate the overall correlation of protein distributions (Fig. 3c–d).”

Nanoclusters of each protein were more likely to be located near the center of synapses than near the edge.

“NCs of each protein were more likely to be located near the center of synapses than near the edge (Fig. 1f, Extended Data Fig. 2i).”

Considering all individual RIM1 localizations, the distance from the nearest pHuse localization decreased as a function of RIM1 δ1.

“Conversely, considering all individual RIM1 localizations, the distance from the nearest pHuse localization decreased as a function of RIM1 δ1 (Fig. 2k).”

Enriched PSD-95 nanoclusters were opposed to RIM1/2 molecule densities that were 2.0 ± 0.1 times the average RIM1/2 synaptic cluster density.

“We found 44.4 ± 3.0 % of PSD-95 NCs to be enriched (Extended Data Fig. 7b), and these NCs were opposed to RIM1/2 molecule densities that were 2.0 ± 0.1 times the average RIM1/2 synaptic cluster density (Fig. 3f).”

GluA2 was significantly enriched relative to RIM1/2 nanoclusters, decaying e-fold per 66.9 ± 15.4 nm.

“Similar to PSD-95, GluA2 was significantly enriched relative to RIM1/2 NCs, decaying e-fold per 66.9 ± 15.4 nm.”

The measured distribution with transsynaptic alignment gained 21.8 ± 0.5% in synaptic strength compared to a uniform distribution of AZ and PSD proteins.

“the measured distribution with transsynaptic alignment gained 21.8 ± 0.5% in synaptic strength compared to a uniform distribution of AZ and PSD proteins (Extended Data Fig. 8), suggesting this synaptic architecture facilitates higher single vesicle response potency.”

• AssumptionRests on an assumption: Comparing the measured transsynaptic alignment to a uniform distribution of active zone and PSD proteins provides a valid estimate of the synaptic strength gain.

RIM1/2 was enriched with respect to Shank nanoclusters.

“and RIM1/2 was correspondingly enriched with respect to Shank NCs (Extended Data Fig. 9b).”

Postsynaptic scaffold molecules farther from the plasma membrane also colocalized with PSD-95 nanoclusters.

“Notably, we found that transsynaptic molecular alignment may extend deeper into the postsynaptic cell, as postsynaptic scaffold molecules farther from the plasma membrane also colocalized with PSD-95 NCs (Extended Data Fig. 9a,c), and RIM1/2 was correspondingly enriched with respect to Shank NCs (Extended Data Fig. 9b).”

44.4 ± 3.0% of PSD-95 nanoclusters were enriched.

“We found 44.4 ± 3.0 % of PSD-95 NCs to be enriched (Extended Data Fig. 7b), and these NCs were opposed to RIM1/2 molecule densities that were 2.0 ± 0.1 times the average RIM1/2 synaptic cluster density (Fig. 3f).”

A similar PSD-95 protein enrichment profile was found relative to the centers of RIM1/2 nanoclusters.

“A similar PSD-95 protein enrichment profile was found relative to the centers of RIM1/2 NCs (Fig. 3e).”

The frequency of action potential-independent spontaneous release events observed in TTX detected with vGpH was similar to the frequency of NMDA receptor-dependent postsynaptic Ca2+ transients measured separately using GCaMP6f.

“The frequency of action potential (AP)-independent spontaneous release events observed in TTX detected with vGpH was similar to the frequency of NMDA receptor (NMDAR)-dependent postsynaptic Ca2+ transients measured separately using the Ca2+ sensor GCaMP6f (Extended Data Fig. 5a).”

Within individual boutons, RIM1 molecules within 40 nm of a pHuse location had significantly higher δ1 than those further away.

“Furthermore, within individual boutons, RIM1 molecules within 40 nm of a pHuse location had significantly higher δ1 than those further away (Fig. 2j).”

The distribution of Bassoon was closer to uniform throughout the synapse.

“Comparisons between these three proteins suggested that Munc13 had a wider distribution than RIM1/2 across the AZ and the distribution of Bassoon was closer to uniform throughout the synapse (Fig. 1g–i, Extended Data Fig. 2f–n).”

Single electrical field stimuli evoked vesicle fusion with a release probability of 0.11 ± 0.01 per bouton.

“Single electrical field stimuli evoked vesicle fusion (Fig. 2a–b, Extended Data Fig. 4a) with a release probability (Pr) of 0.11 ± 0.01 per bouton, comparable to previous measurements, which was also sensitive to extracellular Ca2+ (Extended Data Fig. 4b–d), as expected.”

RIM1/2 staining revealed no change in synaptic cluster size or intensity at any point.

“RIM1/2 staining revealed no change in synaptic cluster size or intensity at any point.”

The measured AZ-PSD distributions showed a significantly elevated pair cross-correlation function compared to simulated AZ-PSD distributions with either distribution fully randomized.

“The measured AZ-PSD distributions showed a significantly elevated PCF compared to simulated AZ-PSD distributions with either distribution fully randomized (Fig. 3b).”

This enrichment was principally dependent on the relative positioning of nanoclusters within synaptic clusters.

“This enrichment was again principally dependent on the relative positioning of NCs within synaptic clusters (Fig. 3e).”

Approximately 72-82% of evoked events arose from single-vesicle fusion.

“By fitting the photon number distributions of evoked and spontaneous events, we estimated that ~72–82% of evoked events arose from single-vesicle fusion (Fig. 2c).”

Near pHuse sites, the average RIM1 δ1 was significantly greater than chance.

“Indeed, near pHuse sites, the average RIM1 δ1 was significantly greater than chance (Fig. 2i).”

3D-STORM imaging of RIM1/2, PSD-95, and GKAP1 at the same synapses confirmed their mutual co-enrichment.

“3D-STORM imaging of RIM1/2, PSD-95, and GKAP1 at the same synapses further confirmed their mutual co-enrichment (Extended Data Fig. 9d–f).”

RIM1/2 localization densities within a 60 nm radius from the centers of PSD-95 nanoclusters were significantly higher than the synaptic cluster average, decaying e-fold per 43.2 ± 12.1 nm away from the peak.

“RIM1/2 localization densities within a 60 nm radius were significantly higher than the synaptic cluster average, decaying e-fold per 43.2 ± 12.1 nm away from the peak.”

RIM1/2 distributions were not affected immediately following the acute NMDA receptor stimulus.

“Unlike PSD-95, RIM1/2 distributions were not affected immediately following the stimulus (Fig. 4d–f).”

Presynaptic nanostructure underwent a strikingly different pattern of reorganization that was detectable only in relation to PSD-95 nanoclusters.

“In contrast, presynaptic nanostructure underwent a strikingly different pattern of reorganization that was detectable only in relation to PSD-95 NCs.”

Quantitatively similar number, characteristics, and alignment of pre- and postsynaptic nanoclusters were found in acute hippocampal slices from adult rats.

“We likewise found quantitatively similar number, characteristics, and alignment of pre- and postsynaptic NCs in acute hippocampal slices from adult rats (Extended Data Fig. 7f–h).”

Munc13 had a wider distribution than RIM1/2 across the active zone.

“Comparisons between these three proteins suggested that Munc13 had a wider distribution than RIM1/2 across the AZ and the distribution of Bassoon was closer to uniform throughout the synapse (Fig. 1g–i, Extended Data Fig. 2f–n).”

There is no significant difference between the two RIM antibodies (anti-RIM1/2 targeting Zn-finger domain and anti-RIM1 targeting PDZ domain) in whole synaptic cluster volume, number of NCs, autocorrelation function estimating average NC diameter, and protein density relative to PSD-95 NC centers.

“These tests suggest that there is no significant difference between these two antibodies.”

Mean distance from pHuse localizations is positively correlated with local density measured by 5 × NND (R=0.23***).

“Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13).”

RIM1/2 is enriched to Shank NCs.

“Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm.”

The isolated non-synaptic localization groups are still significantly smaller than the large majority of detected nanoclusters.

“Nevertheless, we find that they are still significantly smaller than the large majority of the nanoclusters we detected.”

Spontaneous event frequency shows no relationship with Syn1a area.

“spontaneous event frequency showed no relationship with Syn1a area (e, linear fit, evoked R = 0.30**, spontaneous R = 0.12, n.s.)”

Plasticity occurs within nanocolumns.

“Plasticity within nanocolumns”

Cumulative distributions of fusion areas for spontaneous and evoked release differ significantly (K-S test, D=0.23*).

“Cumulative distributions of fusion areas for spontaneous and evoked release (K–S test, D = 0.23*)”

The percentage of PSD-95 NCs enriched with GKAP1, RIM1/2 or both is significantly different from color-coded randomizations.

“The percentage of PSD-95 NCs that were enriched with GKAP1, RIM1/2 or both with color-coded randomizations. *p<0.05, **p<0.01, ANOVA on ranks with pairwise comparison procedures (Dunn’s method), n = 32 NCs from 17 synapses in 7 different cultures.”

There is no difference between PSD-95 clusters labeled with 1°-A647 and 1°-2°-Cy3 in the radius at which the autocorrelation function crossed the random level (g(r)=1).

“There was no difference between PSD-95 clusters with different labeling methods”

Proportion of pHuse localizations within 40 nm of a RIM1 localization is positively correlated with RIM1 local density measured by 5 × NND (R=0.35***).

“Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***).”

RIM1-mEos3.1 PALM identifies NCs.

“RIM1-mEos3.1 PALM identifies NCs”

Nanobodies against GFP conjugated to Atto647 show a significantly smaller size than primary/secondary antibodies against PSD-95.

“The nanobodies showed a significant smaller size than antibodies.”

The enrichment profile between RIM1/2 and PSD-95 in tissue slices is significantly different from randomized synapses (p<0.05).

“Enrichment profile between RIM1/2 and PSD-95 in tissue slices (28 synapses from 7 sections, 4 animals). *p<0.05 between measured and randomized synapses, two way ANOVA with pairwise comparison procedures (Dunn’s method).”

The r(0) for isolated localization groups is significantly less than r(0) for PSD-95 clusters.

“the r(0) for isolated localization groups were significantly less than r(0) for PSD-95 clusters.”

No significant difference was found between evoked area with and without excluding multivesicular events.

“We found no significant difference between evoked area with and without excluding these events (Extended Data Fig. 5d).”

Non-synaptic small groups of localizations from isolated primary antibodies and secondary antibodies show similar variation in all dimensions.

“The two types of localizations groups showed similar variation in all dimensions.”

Homer1 is enriched with PSD-95 NCs.

“Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm.”

pHuse reveals differences between evoked and spontaneous fusion site areas.

“pHuse reveals differences between evoked and spontaneous fusion site areas”

The mixture probability defined the lower estimate (72%) for the percentage of single stimulus evoked fusion arising from single vesicles.

“This mixture probability defined the lower estimate (72%) for the percentage of single stimulus evoked fusion arising from single vesicles.”

• AssumptionRests on an assumption: The number of photons detected in a pHuse event is proportional to the number of vesicles released.

The radius where g(r) first crosses 1 reasonably estimates the average NC diameters.

“Pooled data from 15 sets of simulations showing that the radius where g(r) first crosses 1 reasonably estimates the average NC diameters.”

NMDA induces changes in PSD-95 NC area that are blocked by APV.

“Changes in PSD-95 NC area induced by NMDA and blocked by APV (n = 28 and 21, respectively). **p<0.01, n.s. = not significant, paired t-test.”

Only those RIM1/2 NCs that were enriched with GluR2/3 were increased in volume under control, 5 min NMDA treatment, and 25 min washout conditions.

“Note that similar to the results from the RIM1/2-PSD-95 analyses, only those RIM1/2 NCs that were enriched with GluR2/3 (dark red) were increased in volume.”

The synaptic architecture suggests a simple organizational principle of CNS synapses to maintain and modulate synaptic efficiency.

“This architecture thus suggests a simple organizational principle of CNS synapses to maintain and modulate synaptic efficiency.”

The nanoarchitecture of the active zone directs action potential evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles.

“Thus, we propose that the nanoarchitecture of the active zone directs action potential evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles.”

The precise positioning of the nanoclusters themselves dominates the overall correlation of protein distributions.

“indicating that the precise positioning of the NCs themselves dominate the overall correlation of protein distributions (Fig. 3c–d).”

Because the delayed presynaptic modification was specific to aligned nanoclusters, nanocolumns may point to an alignment-specific, retrograde presynaptic compensation following postsynaptic depression.

“Because the delayed presynaptic modification was specific to aligned NCs, it may be that nanocolumns point to an alignment-specific, retrograde presynaptic compensation following postsynaptic depression (Fig. 4j), potentially relating to previous reports of presynaptic homeostatic plasticity22.”

• cites [22] Davis, G. W. & Müller, M. Homeostatic control of presynaptic neurotransmitter release. Annu. Rev. Physiol. 77, 251–270 (2015) sourceNot checked — no full text of the source

Single synapses have a complex and heterogeneous molecular architecture typified by dense assemblies of fusion-associated proteins nearer the center.

“Together, these observations revealed a complex and heterogeneous molecular architecture within single synapses, typified by dense assemblies of fusion-associated proteins nearer the center.”

The graded protein densities suggest this may not be a clearly delineated structural element.

“The graded protein densities involved suggest this may not be a clearly delineated structural element.”

The detailed metric for assessing nanoscale alignment revealed strong co-enrichment of key proteins along narrow, transcellular columns.

“Thus, this detailed metric for assessing nanoscale alignment revealed strong co-enrichment of these key proteins along narrow, transcellular columns.”

The nanodistribution of RIM predicts the local probability of evoked fusion.

“Thus, nanodistribution of RIM predicts the local probability of evoked fusion.”

The results revealed an axially oriented molecular ensemble spanning the cleft within the bounds of the synapse, evoking the concept of a transsynaptic nanocolumn enriched with key proteins that regulate synaptic transmission.

“Altogether, these results revealed an axially oriented molecular ensemble spanning the cleft within the bounds of the synapse, evoking the concept of a transsynaptic “nanocolumn” enriched with key proteins that regulate synaptic transmission (Extended Data Fig. 9g).”

Similar distribution and nanocluster properties were found using a different antibody targeted toward a separate epitope in RIM1.

“Similar distribution and NC properties were found using a different antibody targeted toward a separate epitope in RIM1 (Extended Data Fig. 2d).”

vGpH increases in green fluorescence intensity upon vesicle fusion.

“vGpH, which increases in green fluorescence intensity upon vesicle fusion12.”

• cites [12] Balaji, J. & Ryan, T. A. Single-vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode. Proc. Natl Acad. Sci. USA 104, 20576–20581 (2007) sourceNot checked — no full text of the source

pHuse area may be a better approximate for AZ area and the functional parameters of a synapse than bouton area.

“This suggests that pHuse area may be a better approximate for AZ area and the functional parameters of a synapse than bouton area.”

The difference between the size of isolated localization groups and PSD-95 clusters calculated by autocorrelation argues against the possibility that the nanoclusters detected were due to repetitive switching of one or a few fluorophores.

“The difference between the size of the isolated localizations groups and PSD-95 clusters calculated by autocorrelation also argues against the possibility that the nanoclusters we detected were due to repetitive switching of one or a few fluorophores.”

The NCs detected in this study were unlikely due to artifacts of antibody binding and labeling.

“Overall, these results demonstrated that the NCs we detected in our study were unlikely due to artifacts of antibody binding and labeling.”

Preferential release in nanocolumns can increase synaptic strength.

“Preferential release in nanocolumns can increase synaptic strength”

Because all transsynaptic analyses were controlled by randomizing simulations, this contamination cannot produce false positives for transsynaptic alignment analyses.

“Since all the transsynaptic analyses were well controlled by randomizing simulations, this contamination is not able to produce false positives for transsynaptic alignment analyses.”

The NCs detected in this study were not due to aggregation of multiple secondary antibodies to the primary antibodies.

“These results demonstrate that the NCs we detected in our study were not due to aggregation of multiple secondary antibodies to the primary antibodies.”

Because the number of localizations in one nanocluster was typically small, using convex hull or alpha shape would greatly under-estimate the nanocluster volume due to the border effect.

“Since the number of localizations in one NC was typically small, using convex hull or alpha shape would greatly under-estimate the NC volume due to the border effect.”

The conservative nanocluster criteria may have prevented detection of small or weakly enriched nanoclusters.

“Consequently, they may have prevented detection of small or weakly enriched NCs.”

The nanocluster identification criteria were chosen conservatively such that no nanoclusters were identified in simulations of randomly distributed synaptic clusters with different densities.

“These criteria were chosen based on a conservative strategy such that no NCs were identified in simulations of randomly distributed synaptic clusters with different densities.”

The contamination may attenuate the significance of differences in transsynaptic analyses based on nanoclusters, including cross-correlation, protein enrichment and the fraction of enriched nanoclusters.

“On the contrary, it may attenuate the significance of the differences in transsynaptic analyses based on NCs, including cross-correlation, protein enrichment and the fraction of enriched NCs.”