Loss of Rab3A prevented the TTX-induced increases in amplitudes and frequency of mEPSCs recorded in cultured mouse cortical neurons.

(A) Ten second example traces recorded at –60 mV in pyramidal cortical neurons from an untreated (“CON”) and TTX-treated (“TTX”) neuron in cultures prepared from Rab3A+/+ mice from the Rab3A+/− colony. (B) Ten second example traces recorded at –60 mV in pyramidal cortical neurons from an untreated (“CON”) and TTX-treated (“TTX”) neuron in cultures prepared from Rab3A−/− mice. (C & D) Average traces for the recordings shown in A and B, respectively. (E) Box plots for average mEPSC amplitudes from untreated cells and TTX-treated cells in cultures prepared from Rab3A+/+ mice (Rab3A+/− colony; CON, N = 30 cells, 13.9 ± 0.7 pA; TTX, N = 23 cells, 18.2 ± 0.9 pA; from 11 cultures). (F) Box plots for average mEPSC amplitudes from untreated cells and TTX-treated cells in cultures prepared from Rab3A−/− mice (CON, N = 25 cells, 13.6 ± 0.7 pA; TTX, N = 26 cells, 14.3 ± 0.6 pA); from 11 cultures. (G) Box plots for average mEPSC frequency for same Rab3A+/+ cells as in (E) (CON, 2.26 ± 0.37 sec−1; TTX, 4.62 ± 0.74 sec−1). (H) Box plots for average mEPSC frequency for same Rab3A−/−cells as in (F) (CON, 2.74 ± 0.49 sec−1; TTX, 3.23 ± 0.93 sec−1). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test. For all p-values, * with underline indicates significance with p < 0.05.

Normally functioning Rab3A was required for TTX-induced homeostatic plasticity of mEPSC amplitudes in cultured mouse cortical neurons.

(A) Ten second example traces recorded at –60 mV in pyramidal cortical neurons from an untreated (“CON”) and TTX-treated (“TTX”) neuron in cultures prepared from Rab3A+/+ mice from the Rab3A+/Ebd colony. (B) Ten second example traces recorded at –60 mV in pyramidal cortical neurons from an untreated (“CON”) and TTX-treated (“TTX”) neuron in cultures prepared from Rab3AEbd/Ebd mice. (C & D) Average traces for the recordings shown in A and B, respectively. (E) Box plots for average mEPSC amplitudes from untreated cells and TTX-treated cells from cultures prepared from Rab3A+/+ mice (Rab3A+/Ebd colony; CON, N = 20 cells, 11.0 ± 0.6 pA; TTX, N = 23 cells, 15.1 ± 1.2 pA; from 6 cultures). (F) Box plots for average mEPSC amplitudes from untreated cells and TTX-treated cells from cultures prepared from Rab3AEbd/Ebd mice (CON, N = 21 cells, 15.1 ± 1.0 pA; TTX, N = 22 cells, 14.6 ± 1.1 pA; from 7 cultures.) (G) Box plots for average mEPSC frequency for same Rab3A+/+ cells as in (E) (CON, 1.15 ± 0.19 sec−1; TTX, 2.54 ± 0.55 sec−1). (H) Box plots for average mEPSC frequency for same Rab3AEbd/Ebd cells as in (F) (CON, 1.71 ± 0.41 sec−1; TTX, 3.05 ± 0.80 sec−1). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test. For all p-values, * with underline indicates significance with p < 0.05.

Homeostatic plasticity of mEPSC amplitudes in mouse cortical cultures treated with TTX for 48 hr was not affected by acute inhibition of Ca2+ permeable AMPA receptors by NASPM (20 μm).

(A) Box plot comparison of the TTX effect on mEPSC amplitudes in the same pyramidal neurons before and after application of 20 μM NASPM. (N = 11 cells from 3 cultures; Pre-NASPM, CON: 12.9 ± 1.1 pA; TTX: 17.5 ± 0.9 pA; post-NASPM, CON: 11.9 ± 0.8 pA; TTX: 16.1 ± 1.0 pA). (B) Line series plot of mEPSC amplitudes before and after acute perfusion with 20 μM NASPM for untreated and TTX-treated pyramidal neurons; same cells as in (A); CON, pre-NASPM: 12.9 ± 1.1 pA; post-NASPM: 11.9 ± 0.8 pA; TTX, pre-NASPM: 17.5 ± 0.9 pA; post-NASPM: 16.1 ± 0.1 pA. (C) Line series plot of mEPSC frequency before and after acute perfusion with 20 μM NASPM for untreated and TTX-treated pyramidal neurons; same cells as in (A); CON, pre-NAPSM: 1.84 ± 0.55 sec−1; post-NASPM; 1.56 ± 0.53 sec−1; TTX, pre-NASPM: 4.40 ± 1.06 sec−1; post-NASPM, 2.68 ± 0.68 sec−1. (D) A proposed mechanism for why NASPM had a robust effect on frequency without greatly affecting amplitude. A dendrite with spines (Top) is expanded on three postsynaptic sites (Middle) to show possible types of AMPA receptor distributions: Left, at a site comprised only of Ca2+-permeable AMPA receptors (NASPM-sensitive, GluA2-lacking receptors (black)), NASPM would completely inhibit the mEPSC, causing a decrease in frequency to be measured in the overall population; Right, at a site comprised only of Ca2+-impermeable AMPA receptors (NASPM-insensitive, GluA2-containing receptors (green)), NASPM would have no effect on mEPSC amplitude; Middle, at a site comprised of a mix of Ca2+-permeable and Ca2+-impermeable AMPA receptors, NASPM would partially inhibit the mEPSC. Diagram produced in BioRender.com (2024). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test. Line series plot p values are from student’s paired t test. For all p-values, * with underline indicates significance with p < 0.05.

Identification of synaptic GluA2 receptor immunofluorescence on primary dendrites of pyramidal neurons in high density mouse cortical co-cultures prepared from Rab3A+/+ mice.

(Top) Non-zoomed single confocal sections collected with a 60X oil immersion objective of recognizably pyramidal-shaped neurons, presumed to be excitatory neurons, selected for synaptic GluA2 analysis. A neuron was selected from an untreated coverslip (CON, Left) and a TTX-treated coverslip (TTX, Right) from the same culture prepared from Rab3A+/+ animals (= Rab3A+/+ Culture #2 in Figure 6). The white rectangular boxes indicate 5X zoomed areas shown in the images below. Note that the depth of the single confocal section of the non-zoomed neuron image is not at the same depth as the confocal section in zoomed dendritic image, so some features are not visible in both images. Scale bar, 20 μm. (Bottom) 5X zoom single confocal sections selected for demonstration purposes because they had an unusually high number of identified synaptic pairs along the primary dendrite contained within a single confocal section. Synaptic pairs, highlighted with white trapezoids, were identified based on close proximity of GluA2 (red) and VGLUT1 (white) immunofluorescence, apposed to the MAP2 immunofluorescent primary dendrite (green). Some apparent synaptic pairs are not highlighted with a white trapezoid because there was a different confocal section in which the two immunofluorescent sites were maximally bright. There were GluA2 positive clusters located on the primary dendrites that were not apposed to VGLUT1-positive terminals (4 of these non-synaptic GluA2 clusters are highlighted with white arrows in the MAP2-GluA2 and MAP2-GluA2-VGLUT1 panels). Scale bar, 10 μm.

Comparison of mEPSC amplitude and GluA2 receptor cluster data, pooled from 3 cortical co-cultures prepared from Rab3A+/+ mice and 3 co-cultures prepared from Rab3A−/− mice.

(A) Mean mEPSC amplitudes recorded in pyramidal neurons from untreated (CON) and TTX-treated coverslips (TTX) of co-cultures prepared from Rab3A+/+ mice (CON, N = 23 cells, 13.7 ± 0.9 pA; TTX, N = 24 cells, 16.4 ± 0.9 pA; from 3 cultures). (B) Mean sizes of synaptic GluA2 receptor clusters, identified by proximity to VGLUT1-positive terminals, on primary dendrites of pyramidal neurons of untreated and TTX-treated coverslips from the same cultures as in (A) (CON, N = 29 dendrites, 0.90 ± 0.06 μm2; TTX, N = 28 dendrites, 1.08 ± 0.10 μm2). (C) Mean intensities of the same GluA2 receptor clusters as in (B) (CON, 684 ± 15 A.U.; TTX, 699 ± 12 A.U.). (D) Mean integrals of the same GluA2 receptor clusters as in (B). (CON, 317,898 ± 26,726 A.U.; TTX, 389,487 ± 41,465 A.U.). (E) Mean mEPSC amplitudes in pyramidal neurons from untreated and TTX-treated coverslips of mouse cortical co-cultures from Rab3A−/− mice (CON, N = 21 cells, 14.9 ± 0.8 pA; TTX, N = 21 cells, 13.5 ± 0.9 pA; from 3 cultures). (F) Mean sizes of GluA2 receptor clusters, identified by proximity to VGLUT1-positive terminals, on primary dendrites of pyramidal neurons of untreated and TTX-treated coverslips from the same cultures as in (E). (CON, N = 30 dendrites, 0.93 ± 0.05 μm2; TTX, N = 30 dendrites, 0.92 ± 0.05 μm2). (G) Mean intensities of the same GluA2 receptor clusters as in (F). (CON, 766 ± 12 A.U.; TTX, 776 ± 78 A.U.). (H) Mean integrals of the same GluA2 receptor clusters as in (F). (CON, 369,436 ± 23,439 A.U.; TTX, 366,389 ± 25,049 A.U.). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10 and 90 percentiles; open circles indicate means of individual cells; line, median; dot, mean. p values (shown on the graphs) were determined with Kruskal-Wallis test. For all p-values, * with underline indicates significance with p < 0.05.

Comparison of mEPSC amplitude and GluA2 receptor cluster characteristics in the same mouse cortical cultures prepared from Rab3A+/+ mice, and another set of cultures prepared from Rab3A−/− mice.

Data pooled from 3 cultures, and data from each of the 3 individual cultures.

In cultures prepared from Rab3A+/+ mice, increases in mEPSC amplitudes following TTX treatment were not always matched by increases in GluA2 receptor cluster characteristics.

(A) Culture #1: mean mEPSC amplitudes for pyramidal neurons from untreated coverslips (CON) and TTX-treated coverslips (TTX) (CON, N = 6, 14.2 ± 2.2 pA; TTX, N = 6, 15.9 ± 1.9 pA), compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken of dendrites from other coverslips in the same culture (CON, N = 10, TTX, N = 9; size, CON, 0.73 ± 0.09 μm2; TTX, 0.89 ± 0.09 μm2; intensity, CON, 682 ± 15 A.U.; TTX, 704 ± 11 A.U.; integral, CON, 256,364 ± 35,242 A.U.; TTX, 316,647 ± 35,077 A.U.). (B) Culture #2: mean mEPSC amplitudes for pyramidal neurons from untreated and TTX-treated coverslips (CON, N = 7, 13.8 ± 2.4 pA; TTX, N = 8, 17.7 ± 1.8 pA), compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken of dendrites from other coverslips from the same culture (CON, N = 9, TTX, N = 9; size, CON, 0.91 ± 0.12 μm2; TTX, 1.42 ± 0.24 μm2; intensity, CON, 621 ± 14 A.U.; TTX, 679 ± 26 A.U.; integral, CON, 296,300 ± 44,108 A.U.; TTX, 512,336 ± 96,892 A.U.). (C) Culture #3: mean mEPSC amplitudes for pyramidal neurons from untreated coverslips and TTX-treated coverslips (CON, N = 10, 13.4 ± 0.8 pA; TTX, N = 9, 15.7 ± 1.0 pA,) compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken from other coverslips from the same culture. (CON, N = 10, TTX, N = 9; size, CON, 1.05 ± 0.11 μm2; TTX, 0.95 ± 0.15 μm2; intensity, CON, 744 ± 28 A.U.; TTX, 714 ± 25 A.U.; integral, CON, 398,870 ± 49,500 A.U.; TTX, 347,571 ± 65,454 A.U.). Cultures were fixed and processed for immunofluorescence on the same day as mEPSC recordings, and imaged within 1 week. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test. p-values denoted with * and underline indicates significance of p < 0.05.

In cultures prepared from Rab3A−/− mice, a lack of effect of TTX treatment in mEPSC amplitudes was not always matched by a lack of effect in synaptic GluA2 receptor cluster characteristics.

(A) Culture #1: mean mEPSC amplitudes for pyramidal neurons from untreated coverslips (CON) and TTX-treated coverslips (TTX) (CON, N = 7, 15.4 ± 1.2 pA; TTX, N = 6, 14.4 ± 0.8 pA), compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken of dendrites from other coverslips in the same culture (CON, N = 10, TTX, N = 10; size, CON, 0.87 ± 0.07 μm2; TTX, 0.80 ± 0.12 μm2; intensity, CON, 735 ± 10 A.U.; TTX, 731 ± 25 A.U.; integral, CON, 328,477 ± 28,250; TTX, 302,594 ± 53,052 A.U.). (B) Culture #2: mean mEPSC amplitudes for pyramidal neurons from untreated and TTX-treated coverslips (CON, N = 8, 15.2 ± 1.8 pA; TTX, N = 9, 13.5 ± 1.8 pA), compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken of dendrites from other coverslips in the same culture (CON, N = 10, TTX, N = 10; size, CON, 1.05 ± 0.10 μm2; TTX, 0.93 ± 0.05 μm2; intensity, CON, 800 ± 26 A.U.; TTX, 781 ± 21 A.U.; integral, CON, 425,552 ± 49,486 A.U.; TTX, 361,740 ± 21,963 A.U.). (C) Culture #3: mean mEPSC amplitudes for pyramidal neurons from untreated and TTX-treated coverslips (CON, N = 6, 13.9 ± 1.2 pA; TTX, N = 6, 12.9 ± 1.5 pA) compared to mean sizes, intensities, and integrals of synaptic GluA2 receptor clusters in images taken of dendrites from other coverslips in the same culture (CON, N = 10, TTX, N = 10; size, CON, 0.89 ± 0.08 μm2; TTX, 1.03 ± 0.08 μm2; intensity, CON, 765 ± 22 A.U.; TTX, 814 ± 23 A.U.; integral, CON, 354,279 ± 38,755 A.U.; TTX, 434,833 ± 42,350 A.U.). Cultures were fixed and processed for immunofluorescence on the same day as mEPSC recordings, and imaged within 1 week. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test.

Comparison of mEPSC amplitude and VGLUT1 positive presynaptic site characteristics in the same mouse cortical cultures prepared from Rab3A+/+ and Rab3A−/− mice.

Data pooled from 3 cultures, and data from individual cultures. mEPSC data are identical to that in Table 1, reproduced here for comparison purposes.

Rab3A in neurons, not astrocytes, was required for full TTX-induced homeostatic plasticity.

(A)-(C), mEPSC amplitude (Middle) and frequency (Right) data from dissociated cortical neurons plated on an astrocyte feeder layer, each prepared separately from the type of mice depicted in the schema (Left): (A) Neurons from Rab3A+/+ mice plated on astrocytes from Rab3A+/+ mice.

Box plots for mEPSC amplitudes (CON, N = 17 cells, 13.3 ± 0.5 pA; TTX, N = 20 cells, 16.7 ± 1.2 pA) and mEPSC frequency in the same recordings (mean, CON, 2.54 ± 0.57 sec−1; TTX, 3.48 ± 0.64 sec−1); from 4 cultures. (B) Neurons from Rab3A+/+ mice plated on astrocytes from Rab3A−/− mice. Box plots for average mEPSC amplitude (CON, N = 11 cells, 13.3 ± 1.0 pA; TTX, N = 11 cells, 18.8 ± 1.4 pA) and average mEPSC frequency in the same recordings (CON, 2.01 ± 0.41 sec−1; TTX, 4.47 ± 1.53 sec−1); from 2 cultures. (C) Neurons from Rab3A−/− neurons plated on astrocytes from Rab3A+/+ mice. Box plots for average mEPSC amplitude (CON, N = 14 cells, 15.2 ± 1.1 pA; TTX, N = 11 cells, 16.9 ± 0.7 pA) and mEPSC frequency in the same recordings (CON, 4.47 ± 1.21 sec−1; TTX, 3.02 ± 0.70 sec−1); from 3 cultures. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; line, median; dot, mean. p values (shown on the graphs) are from Kruskal-Wallis test. p-values denoted with * and underline indicates significance of p < 0.05.

Model depicting the requirement for neuronal Rab3A in the homeostatic increase in synaptic GluA2 receptor levels after treatment with TTX.

Under normal activity levels (Left), neither presynaptic or postsynaptic Rab3A is engaged in the regulation of GluA2 receptor levels. After prolonged activity blockade (Right), Rab3A located in the presynaptic terminal promotes the release of a trophic factor (orange dots) that diffuses across the synapse and facilitates transport of postsynaptic vesicles containing GluA2 AMPA receptors (dark orange channels) to the surface membrane. Rab3A located in the postsynaptic dendrite directly promotes fusion of vesicles containing GluA2 AMPA receptors. Fusion of presynaptic vesicles containing glutamate (black dots) causes mEPSCs when glutamate binds to AMPA receptors. Adapted from “Synaptic Cleft” template, by BioRender.com (2024).