Neuroscience

Molecular and organizational diversity intersect to generate functional synaptic heterogeneity within and between excitatory neuronal subtypes

A. T. Medeiros
S.J. Gratz author has email address
A. Delgado
J.T. Ritt
Kate M. O’Connor-Giles author has email address

Neuroscience Graduate Training Program, Brown University, Providence, RI
Department of Neuroscience, Brown University, Providence, RI
Carney Institute for Brain Science, Brown University, Providence, RI

https://doi.org/10.7554/eLife.88412.1

Open access
Copyright information

Figures and data

VGCC levels predict P_r within, but not between, synaptic subtypes.
(A) Representative confocal Z-projection of Cac^Td-Tomato-N (magenta) with type Ib terminals (blue) and type Is (red) outlined. (B) AZ heat map of terminals in A with color indicating release probability (P_r) and size representing sum Cac intensity levels in arbitrary units (AU). (C) Average single-AZ probability of release at type Ib and type Is terminals. (D) Quintile distribution of single-AZ P_r frequency at type Ib and type Is inputs. (E, F) Correlation between normalized Cac^Td-Tomato-N intensity and P_r at type Is and Ib AZs in the same 6 NMJs. Each dot represents a single AZ and each color corresponds to an individual NMJ with linear regression lines indicated for each NMJ. (G) Top, representative confocal Z-projection of Cac^sfGFP-N. Bottom, Cac^sfGFP-N in green with HRP marking neuronal membranes in gray. Type Ib (blue) and type Is (red) terminals are outlined. (H) Quantification of Cac^sfGFP-N AZ intensity levels at type Ib and type Is terminals. Each data point represents the average normalized single AZ sum intensity for an individual NMJ. (I) Distribution of normalized Cac^sfGFP-N intensity from single type Ib and type Is AZs in H (X-axis cutoff at 5.0). (J) Correlation between normalized Cac^Td-Tomato-N and P_r across all Ib and Is AZs combined from E-F with linear regression lines (blue and red, respectively) and 95% confidence intervals (black lines) indicated. All scale bars = 5µm.

VGCC clusters are more compact at AZs of high-Pr type Is inputs.
(A-C) Representative SoRa Z-projection of Cac^HaloTag-N (green), Brp (magenta), and merge. (D, E) Representative boutons of STORM Cac^HaloTag-N clusters as identified by DBSCAN at type Ib and type Is boutons as indicated. Each color represents an individual identified cluster with purple scattered dots identifying excluded background signal. (F) Quantification of Cac^HaloTag-N cluster area at type Ib and type Is AZs. (G) Quantification of localizations per cluster at type Ib and type Is boutons. (H) Calculated Cac^HaloTag-N cluster density at type Ib and Is AZs. For F-H, each data point represents the respective single-cluster measurement averaged over individual boutons. (I) A paired analysis of calculated AZ cluster density averaged over individual type Ib and Is inputs at the same NMJ. All scale bars = 1µm.

Differences in Bruchpilot levels and function at low-and high-Pr inputs.
(A) Representative confocal Z-projection of Brp expression at type Ib (blue outline) and type Is (red outline) terminals. (B) Quantification of Brp intensity levels at type Ib and type Is AZs. (C) Ratio of normalized Cac^sfGFP-N:Brp levels at type Ib and type Is NMJs. (D) Correlation of Cac^sfGFP-N and Brp at type Ib and type Is single AZs with linear regression lines (blue and red, respectively) and 95% confidence intervals (black dotted lines) indicated. (E, F) Representative confocal Z-projections of Cac^sfGFP-N (green), Brp (magenta), HRP (white), and merge at type Ib (blue outline) and Is (red outline) terminals of Cac^sfGFP-N(WT) or Cac^sfGFP-N;brp^-/- (brp^-/-) animals. (G) Quantification of Cac^sfGFP-N normalized fluorescence intensity levels at type Ib and type Is AZs of WT vs brp^-/- animals. (H) Ratio of Cac^sfGFP-N fluorescence intensity levels at type Ib and type Is AZ in brp^-/-:WT. For B and G, each data point represents the normalized single AZ sum intensity measurements averaged over individual NMJs. All scale bars = 5µm.

Brp differentially regulates VGCC dynamics at low-and high-Pr inputs during presynaptic homeostatic potentiation.
(A, B) Representative confocal Z-projections of Cac^sfGFP-N (top, green), Brp (middle, magenta) and merged with HRP (bottom, gray) in untreated and PhTx-treated Cac^sfGFP-N NMJs showing type Ib (blue) and type Is (red) terminals. (C) Quantification of Brp fluorescence intensity levels. (D) Quantification of Cac^sfGFP-N fluorescence intensity levels. (E, F) Representative confocal Z-projections of Cac^sfGFP-N (top, green), Brp (middle, magenta) and merged with HRP (bottom, gray) in untreated and PhTx-treated Cac^sfGFP-N;brp^-/- NMJs showing type Ib (blue) and type Is (red) terminals. (G) Quantification of Cac^sfGFP-N fluorescence intensity levels. For all quantifications, each data point represents normalized single AZ sum intensity measurements averaged over individual NMJs. All scale bars = 5µm.

Endogenous tagging of VGCC auxiliary subunits reveals distinct synaptic expression patterns.
(A) Schematic of Ca²⁺ channel complex with tagged auxiliary subunits (created with BioRender). (B) Schematic of Ca-β (isoform PL shown), Stj (isoform PC), and Stolid (isoform H/I) endogenous tag locations. (C-E) Quantifications of EJPs, mEJPs, and quantal content for each endogenously tagged line. (F-H) Representative confocal Z-projections of auxiliary subunit expression (green) at the larval ventral ganglion (VG, top, scale bars = 100µm), and NMJs co-labeled with anti-HRP (magenta, middle and bottom, scale bars = 5µm).

Stj/α2δ-3 levels are lower at AZs of high-P_r type Is inputs.
(A) Representative SoRa Z-projections of Ca-β^V5-C (green), Brp (magenta) and merge. (B) Representative SoRa Z-projections of Cac^sfGFP-N (green), Stj^V5-N (magenta), and merge. Scale bars for A and B = 1μm. (C, D) Representative confocal Z-projections of Ca-β^V5-C expression and Stj^V5-N expression at type Ib (blue outline) and type Is (red outline) NMJs. Scale bar = 5μm. (E, F) Quantification of Ca-β^V5-C and Stj^V5-N fluorescence intensity levels at type Ib and type Is AZs. Each data point represents normalized single AZ sum intensity measurements averaged over individual NMJs. (G, H) Correlation of Cac^sfGFP-N and Stj^V5-N fluorescence intensity levels at type Ib and type Is single AZs with linear regression lines (color lines) and 95% confidence intervals (black lines).

Absolute values and statistics.
All comparisons, Ns (animals, NMJs, AZs (AZs), and statistical tests used in this study. All values are mean ± SEM.

Absolute values and statistics.
All comparisons, Ns (animals, NMJs, AZs (AZs), and statistical tests used in this study. All values are mean ± SEM.

Imaging details.
This table contains detailed information on how each protein was labeled and visualized using live and fixed confocal microscopy and STORM imaging. All secondary antibodies were incubated at RT for 2 hours at a concentration of 1:500.

Imaging details.
This table contains detailed information on how each protein was labeled and visualized using live and fixed confocal microscopy and STORM imaging. All secondary antibodies were incubated at RT for 2 hours at a concentration of 1:500.

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