A trans-synaptic nanocolumn aligns neurotransmitter release to receptors

In the paper

“However, observations in small CNS synapses of both clustered24,25 and random distribution of Ca2+ channels26, and emerging evidence for channel mobility as an equalizer of Pr for vesicles independent of channel positioning27 suggest that their precise distribution may not be the sole determinant of the AZ release likelihood landscape.”

What the sources say

• Disputed

  Split into 2 atoms — 0 confirmed, 0 partially supported, 2 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  cites [24] Holderith, N. et al. Release probability of hippocampal glutamatergic terminals scales with the size of the active zone. Nat. Neurosci. 15, 988–997 (2012) sourceExperimental — accuracy not yet measured
• Disputed

  Split into 2 atoms — 1 confirmed, 0 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  cites [25] Nakamura, Y. et al. Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development. Neuron 85, 145–158 (2015) sourceExperimental — accuracy not yet measured
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [26] Scimemi, A. & Diamond, J. S. The number and organization of Ca2+ channels in the active zone shapes neurotransmitter release from Schaffer collateral synapses. J. Neurosci. 32, 18157–18176 (2012) source
• ?Not checked — no full text of the sourcecites [27] Schneider, R. et al. Mobility of calcium channels in the presynaptic membrane. Neuron 86, 672–679 (2015) source

In the paper

“We next tested an acute 5-minute activation of NMDARs, known to induce a sustained depression of synaptic strength20,21.”

What the sources say

• ?Not checked — no full text of the sourcecites [20] Lee, H.-K., Kameyama, K., Huganir, R. L. & Bear, M. F. NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus. Neuron 21, 1151–1162 (1998) source
• Supported

  The Results section (page unavailable) says brief NMDA application produces chemical long-term depression. It also reports that a 5-minute NMDA treatment removed GluA1 AMPA receptors from synapses in wild-type neurons, with the reduction persisting for at least 30 minutes. This supports the paraphrase, although the 5-minute experiment measured receptor localization rather than synaptic strength directly. (LLM, conf=0.87)

  cites [21] Sanderson, J. L. et al. AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors. J. Neurosci. 32, 15036–15052 (2012) source

In the paper

“given that the probability of AMPAR activation declines with distance from glutamate release sites has previously been deduced3,16”

What the sources say

• Supported

  In “Dependence on release location,” the paper reports that the open probability of an individual AMPA receptor decreased as its distance from the glutamate release site increased. Figure 7C in “Implications of RLD” likewise describes a decreasing average probability of receptor activation with increasing radial distance from release. (LLM, conf=0.95)

  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) source
• ?Not checked — no full text of the sourcecites [16] Raghavachari, S. & Lisman, J. E. Properties of quantal transmission at CA1 synapses. J. Neurophysiol. 92, 2456–2467 (2004) source

In the paper

“The distribution of RIM1-mEos3 was nonuniform and contained NCs with an average diameter of 80.95 ± 5.34 nm and 78.93 ± 5.85 nm using either an adapted SR-Tesseler analysis15 or nearest neighbor distance analysis4, respectively (Extended Data Fig. 6f), consistent with our 3D-STORM results (Fig. 1).”

What the sources say

• ?Not checked — no full text of the sourcecites [15] Levet, F. et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat. Methods 12, 1065–1071 (2015) source
• Partly supported

  In Results (‘Scaffold molecules are organized in subsynaptic clusters’; page unavailable), the source reports postsynaptic scaffold clusters with an 83 ± 4 nm diameter, broadly consistent with the cited sentence’s nanoscale-cluster comparison. It does not report RIM1-mEos3 measurements or either stated diameter, and its described analyses count neighbors within a radius and use pair-correlation and Monte Carlo methods rather than explicitly identifying a nearest-neighbor distance analysis. (LLM, conf=0.82)

  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) sourceExperimental — accuracy not yet measured

In the paper

“To test this, we induced long-term potentiation via APV withdrawal and glycine stimulation19 which resulted in an increase in PSD-95 localization density within NCs, the percentage of PSD-95 NCs enriched with RIM1/2, and the enrichment index of PSD-95 NCs (Fig. 4a–c, Extended Data Fig. 10m).”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  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) sourceExperimental — accuracy not yet measured

In the paper

“GCaMP6f was used to detect postsynaptic miniature spontaneous Ca2+ transients (mSCaTs) that arose in dendritic spines following NMDA receptor activation by spontaneous release46.”

What the sources say

• Partly supported

  The Abstract reports fluorescence detection of NMDA receptor-mediated miniature calcium transients caused by spontaneous vesicle release in dendrites. But Methods > Calcium Imaging specifies fluo-3, not GCaMP6f, and the Discussion says the authors generally could not resolve spines; it does not establish that the observed transients arose in spines. (LLM, conf=0.91)

  cites [46] Murthy, V. N., Sejnowski, T. J. & Stevens, C. F. Dynamics of dendritic calcium transients evoked by quantal release at excitatory hippocampal synapses . Proc. Natl Acad. Sci. USA 97, 901–906 (2000) sourceExperimental — accuracy not yet measured

In the paper

“TetraSpeck beads (100 nm; Invitrogen) deposited on a coverslip were localized to correct alignment between the two channels as described4. The average deviation of the bead localizations after correction was between 10 and 15 nm.”

What the sources say

• Partly supported

  The Experimental Procedures (page unavailable) say that images from two channels were acquired simultaneously, drift-corrected, and aligned. Our copy does not say that TetraSpeck beads were used for alignment or report a 10–15 nm average deviation after correction; the Results’ <15 nm figure concerns molecule-localization precision, not bead alignment. (LLM, conf=0.84)

  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) sourceExperimental — accuracy not yet measured

In the paper

“Syn1a-CFP puncta area calculated as a measure of bouton area, which correlated with pHuse area, as expected42.”

What the sources say

• Partly supported

  The Serial electron microscopy section reports that postsynaptic density area was proportional to presynaptic axonal varicosity volume, supporting a general relationship between bouton size and synaptic size. It does not report Syn1a-CFP puncta, pHuse area, or a correlation between those two fluorescence measurements. (LLM, conf=0.82)

  cites [42] Harris, K. M. & Stevens, J. K. Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982–2997 (1989) sourceExperimental — accuracy not yet measured

In the paper

“Different from the ACF, overcounting has no effect on the PCF39.”

What the sources say

• Partly supported

  In Materials and Methods (“Modifications for measured cross-correlation functions”; page unavailable), the paper says pair cross-correlation functions have no over-counting artifact, unlike pair auto-correlation functions. It limits this result to labeling schemes in which two distinguishable probes cannot label the same molecule; the unqualified claim omits that condition. (LLM, conf=0.82)

  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) sourceExperimental — accuracy not yet measured

In the paper

“we found that action potential evoked fusion was guided by this protein gradient and occurred preferentially in confined areas with higher local density of RIM within the active zones.”

In the paper

“which was also sensitive to extracellular Ca2+ (Extended Data Fig. 4b–d), as expected.”

In the paper

“An example synapse, presented in Fig. 3a (suppl. Video 1), shows three RIM1/2 NCs and three PSD-95 NCs that appear well-aligned and one pair not aligned.”

In the paper

“In fact, evoked fusion site areas were significantly smaller (median smaller by 48%) and occurred over a significantly smaller proportion of the bouton (median smaller by 39%) than spontaneous fusion (Fig. 2g, Extended Data Fig. 5c–d, h–j).”

In the paper

“In comparison to RIM1/2, the enrichment of Munc13 with respect to PSD-95 NCs was considerably weaker, and Bassoon intermediate (Fig. 3d, g, Extended Data Fig. 7c–e, Suppl. Table 2).”

In the paper

“Fusion site areas for spontaneous and evoked vesicle fusion tightly correlated with bouton areas measured by Syn1a (Fig. 2f), as expected.”

In the paper

“However, the slopes of the correlations differed, even though the bouton sizes were similar between groups (Extended Data Fig. 5b).”

In the paper

“Compared to PSD-95 as the common control in pairwise two-color experiments, there were similar numbers of RIM1/2, more Munc13, and fewer Bassoon NCs per synapse (Fig. 1h).”

In the paper

“These effects were long-lasting, and during the subsequent 25 minutes, most parameters underwent only partial recovery.”

In the paper

“Remarkably, while RIM1/2 NCs altogether remained constant in number and enriched percentage, there was in fact an increase in the size of those RIM1/2 NCs that were enriched with PSD-95, whereas the other non-enriched RIM1/2 NCs remained constant (Fig. 4i).”

In the paper

“Notably, no changes in RIM1/2 were observed, consistent with LTP as a primarily postsynaptic phenomenon.”

In the paper

“Together, both the PCFs and protein enrichment analyses revealed significant trans-synaptic alignment between RIM1/2 and PSD-95 distributions, largely stemming from the correlated positions of their respective NCs.”

In the paper

“which showed that release events preferentially occurred in regions with normalized RIM local density greater than 1.5, and these events occurred over 20–60% of the AZ area”

In the paper

“We calculated the higher estimate (82%) by calculating the percent of evoked fusion events with photon counts within two standard deviations of the mean spontaneous fusion event photon count.”

In the paper

“Comparison of NC number, fraction of localization in NC, and NC volume across different developmental stages shows no significant difference, though the young DIV9 culture shows a trend toward increased NC numbers”

In the paper

“This alpha shape algorithm gave a synaptic cluster volume of 81 ± 3 % of the convex hull volume (n = 156 synapses).”

In the paper

“Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons.”

In the paper

“The fraction of enriched NCs is significantly above chance level, and is also dependent on the relative position of the two sets of NCs.”

In the paper

“NCs detected in both channels displayed no difference in number, volume, or the fraction of NCs enriched with localizations from the other channel.”

In the paper

“Normalized pHuse area was not significantly different at different thresholds for Syn1a within modes of release but still significantly different between modes of release (n = 51/10).”

In the paper

“Normalized pHuse area as a function of cell age shows no significant correlation (evoked R = 0.03, n.s., spont R = 0.004, n.s.)”

In the paper

“Paired autocorrelation function of synaptic clusters with and without multiple-frame molecules. p = 0.77, n = 25 synapses for RIM1/2; p = 0.58, n = 25 synapses for PSD-95, two-way ANOVA with repeated measures.”

In the paper

“Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells).”

In the paper

“An additional criterion to exclude evoked pHuse localizations with photon counts > mean + 2SD of spontaneous photon count distribution was used in Extended Data Fig. 5d and showed no significant difference compared to the distribution lacking this criterion.”

In the paper

“There were, on average, 2.02 ± 0.08 and 1.32 ± 0.21 NCs with a volume of (3.6 ± 0.2) and (4.2 ± 0.7) × 105 nm3 for RIM1/2 and PSD-95, respectively.”

In the paper

“Both proteins showed significant enrichment in PSD-95 NCs, *p<0.05, paired t-tests.”

In the paper

“The tracking removed 13 ± 8% and 17 ± 9% of the localizations for RIM1/2 and PSD-95, respectively, but had no significant effects on autocorrelation function results, NC numbers, or NC volumes.”

In the paper

“Both numbers of events and mode of release are significant factors for pHuse area, but they do not have a significant interaction nevoked = 155/38, nspont = 109/29.”

In the paper

“Cumulative distributions of normalized fusion areas for 1 AP evoked fusion excluding events with photon counts > mean + 2SD of spontaneous events (n = 91/27) compared to all evoked events (n = 104/28, K–S test, D = 0.05, n.s.) and spontaneous events (n = 77/22, K–S test, D = 0.25*)”

In the paper

“the calculated synaptic response was independent of pixilation level for sampling size from 1 to 20 nm, data not shown”

In the paper

“Comparison of spontaneous frequency measured presynaptically using vGpH (n = 77/22) and postsynaptically using GCaMP6f 45(n = 61/5), t = 1.02, n.s.”

In the paper

“Nanoclusters identified by both methods were similar in size (Extended Data Fig. 6).”

In the paper

“both spontaneous event frequency and evoked Pr significantly positively correlated with pHuse area (f, linear fit, evoked R = 0.64***, spontaneous R = 0.60***)”

In the paper

“Except PSD NC number which was significantly less than that in cultures (p = 0.03), all other parameters were similar (Wilcoxon signed-rank test).”

In the paper

“1AP evoked release is [Ca2+] dependent and mainly univesicular48”

What the sources say

• ?Not checked — no full text of the sourcecites [48] Kavalali, E. T. et al. Spontaneous neurotransmission: an independent pathway for neuronal signaling? Physiology (Bethesda) 26, 45–53 (2011) source

In the paper

“33% and 67% thresholds were also compared and showed no significant difference on the effect of mode of release, shown in Extended Data Fig. 5i”

In the paper

“Similar detection thresholds were set for spontaneous (75 ± 15) and evoked (78 ± 14, t = 0.88, p = 0.40) release, at ~3–4 times above background noise, on an individual imaging field basis.”

In the paper

“Normalized pHuse area was not significantly different at RT (nevoked = 51/10, nspont = 32/7) vs physiological temperature (nevoked = 35/9, nspont = 34/4) within modes of release but still significantly different between modes of release.”

In the paper

“Average bouton areas across groups, t = 0.87, n.s.”

In the paper

“LTP stimulation induced changes in NC volumes (l), localization density within NCs (m) and NC numbers (n). *p<0.05, ANOVA on ranks with pairwise comparison to control group (Dunn’s method).”

In the paper

“The compartmentalized AZ architecture is reminiscent of protein organization in Drosophila neuromuscular junction23 and vertebrate ribbon synapses, where vesicles and priming proteins are arrayed around tight clusters of Ca2+ channels.”

What the sources say

• ?Not checked — no full text of the sourcecites [23] Liu, K. S. et al. RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334, 1565–1569 (2011) source

In the paper

“Overall, the gradients of protein density we observed suggest a nanocolumn model, in which AZ regions with the highest likelihood of release are aligned to the densest receptor areas, optimizing the potency of neurotransmission (Suppl. Video 2).”

In the paper

“This predicts that pHuse events would be associated with higher local RIM1 density and conversely that high local density of RIM1 increases the probability of nearby fusion.”

In the paper

“This and other experiments suggest that the measured nonuniformity was not likely due to over-counting molecules or to potential artifacts of primary-secondary antibody labeling (Extended Data Fig. 3).”

In the paper

“This provides a simple organizational principle that may hold for many small, CNS synapses, and will have the largest influence at synapses that typically release only one vesicle following an AP.”

In the paper

“For comparison, long-term depression induces a very similar magnitude decrease in synaptic strength18.”

What the sources say

• ?Not checked — no full text of the sourcecites [18] Dudek, S. M. & Bear, M. F. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-d-aspartate receptor blockade. Proc. Natl Acad. Sci. USA 89, 4363–4367 (1992) source

In the paper

“This was further confirmed with a different GluA2/3 antibody (Suppl. Table 2).”

In the paper

“One interpretation is that the concentration of vesicle priming proteins in NCs favors evoked fusion in these subregions of the AZ.”

In the paper

“Homogeneity of variances was tested using an F-test and found to be similar between compared groups.”

In the paper

“For a uniform distribution, e.g. when all localizations were uniformly randomized within the alpha shape, ga(r) = 1 (Fig. 1d). Any heterogeneity will result in a ga(r) > 1.”

In the paper

“If localization assemblies inside the two synaptic clusters organized in a similar pattern and opposed each other, gc(r) > 1. If either synaptic cluster had a uniform distribution of localizations (Fig. 3b) or the internal assemblies were not aligned (Fig. 3c), gc(r) = 1.”

In the paper

“Since the information of synaptic cluster shape and overall density had been normalized, gc(r) was fully dependent on the internal organizations of the two synaptic clusters.”

In the paper

“This equation is derived from fitting MdNND with d in a series of simulations of uniformly distributed synaptic clusters with different densities.”

In the paper

“By rotating a 3D scatterplot of localizations of a selected potential synapse, we evaluated the data quality and selected only those with clear pre- and postsynaptic components (e.g. no nearby third cluster which may indicate two synapses in close proximity) for further analysis.”

In the paper

“All experimental protocols were approved by the University of Maryland, Baltimore School of Medicine Institutional Animal Care and Use Committee.”

In the paper

“For comparison of two or more distributions, all samples were assessed for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests.”

In the paper

“Single molecule tracking was employed to remove the overcounted localizations from peaks lasting for more than one frame.”

In the paper

“This value was determined based on series of tests on >100 synapses to obtain the best synaptic cluster shape while avoiding dramatic changes in volume when individual localizations near the border were added or removed.”

In the paper

“In principal, we cannot completely exclude the possibility of overcounting, so a certain fraction of detected NCs are potentially artificial.”

In the paper

“The sample sizes were determined based on numbers reported in previous studies.”

In the paper

“No statistical methods were used to predetermine sample size.”

In the paper

“The average deviation of localized z-positions of immobilized fluorophores was 40–50 nm.”

In the paper

“All experiments were performed on neurons 14–21DIV and repeated on 3 or more separate cultures unless otherwise specified.”

In the paper

“Only molecules localized with an x-y precision <10 nm37, fitting R2 > 0.6, and comprising >200 photons were used for further analysis.”

What the sources say

• ?Not checked — no full text of the sourcecites [37] Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002) source

In the paper

“These localization groups likely represent an overestimate of a single-dye-molecule localization spread.”

In the paper

“A synaptic cluster was only considered for analysis if the volume was between 2 × 10−3 μm3 and 30 × 10−3 μm3 (Cit.38), and contained an average density of > 8 × 103 localizations/μm3.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Cit.38