Homeostatic synaptic plasticity of miniature excitatory postsynaptic currents in mouse cortical cultures requires neuronal Rab3a
- Department of Pharmacology, Physiology, and Neurobiology, University of Cincinnati College of Medicine, United States
- Department of Neuroscience, Cell Biology and Physiology, Boonshoft School of Medicine, Wright State University, United States
- Department of Neuroscience, Cell Biology and Physiology, Boonshoft School of Medicine and the College of Science and Mathematics, Wright State University, United States
Figures
Figure 1
Loss of Rab3a disrupted the TTX-induced increase in amplitudes of miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured mouse cortical neurons; the increase in frequency was more variable but also appears to be reduced.
(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 eleven 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 eleven cultures. (G) Box plots for average mEPSC frequency for same Rab3a+/+ cells as in (E) (CON, 2.26 ± 0.37 s–1; TTX, 4.62 ± 0.74 s–1). (H) Box plots for average mEPSC frequency for same Rab3a−/− cells as in (F) (CON, 2.74 ± 0.49 s–1; TTX, 3.23 ± 0.93 s–1). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells, bin size 0.1 pA, 0.5 s–1; line, median; dot, mean. p-values (shown on the graphs) are from Tukey’s post hoc test following a two-way ANOVA. For all p-values, * with underline indicates significance with p < 0.05.
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Figure 1—source data 1
Source data used in Figure 1 to show loss of TTX effect in Rab3a-/- mouse cortical neuron cultures.
- https://cdn.elifesciences.org/articles/90261/elife-90261-fig1-data1-v1.xlsx
Figure 2
Normally functioning Rab3a was required for TTX-induced homeostatic plasticity of miniature excitatory postsynaptic current (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 six 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 seven cultures). (G) Box plots for average mEPSC frequency for same Rab3a+/+ cells as in (E) (CON, 1.15 ± 0.19 s–1; TTX, 2.54 ± 0.55 s–1). (H) Box plots for average mEPSC frequency for same Rab3aEbd/Ebd cells as in (F) (CON, 1.71 ± 0.41 s–1; TTX, 3.05 ± 0.80 s–1). Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; bin size 0.1 pA, 0.5 s–1; line, median; dot, mean. p-values (shown on the graphs) are from a Tukey’s post hoc test following a two-way ANOVA. For all p-values, * with underline indicates significance with p < 0.05.
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Figure 2—source data 1
Source data for Figure 2 showing loss of TTX effect on mEPSC amplitude in Rab3a Ebd/Ebd cortical neuron cultures.
- https://cdn.elifesciences.org/articles/90261/elife-90261-fig2-data1-v1.xlsx
Figure 3
Homeostatic plasticity of miniature excitatory postsynaptic current (mEPSC) amplitudes in mouse cortical cultures treated with TTX for 48 hr was unchanged 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 three 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-NASPM: 1.84 ± 0.55 s–1; post-NASPM; 1.56 ± 0.53 s–1; TTX, pre-NASPM: 4.40 ± 1.06 s–1; post-NASPM, 2.68 ± 0.68 s–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 was created using BioRender.com. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; open circles represent means from individual cells; bin size 0.1 pA; 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.
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Figure 3—source data 1
Source data for effects of NASPM on untreated and TTX-treated cortical neuron cultures.
- https://cdn.elifesciences.org/articles/90261/elife-90261-fig3-data1-v1.xlsx
Figure 4
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 5). 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 the 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 (four of these non-synaptic GluA2 clusters are highlighted with white arrows in the MAP2-GluA2 and MAP2-GluA2-VGLUT1 panels). Scale bar, 10 µm. Images have been enhanced for visualization purposes only. No image manipulation was performed prior to signal quantification.
Figure 5 with 1 supplement
Comparison of miniature excitatory postsynaptic current (mEPSC) amplitudes and GluA2 receptor cluster areas in matched and unmatched mouse cortical cultures prepared from Rab3a+/+ mice and treated with TTX for 48 hr.
(A) Culture averages of mEPSC amplitudes for untreated (CON) and TTX-treated coverslips (TTX) in each of three Rab3a+/+ mouse cortical co-cultures. Culture #1, CON, N = 6, 14.2 ± 2.2 pA; TTX, N = 6, 15.9 ± 1.9 pA; Culture #2, CON, N = 7, 13.8 ± 2.4 pA; TTX, N = 8, 17.7 ± 1.8 pA; Culture #3, CON, N = 10, 13.4 ± 0.8 pA; TTX, N = 9, 15.7 ± 1.0 pA. (B) Culture averages of GluA2 receptor cluster size in the same three cultures as shown in (A). Culture #1, CON, N = 10, 0.73 ± 0.09 µm2; TTX, N = 9, 0.89 ± 0.09 µm2; Culture #2, CON, N = 9, 0.91 ± 0.12 µm2; TTX, N = 9, 1.42 ± 0.24 µm2; Culture #3, CON, N = 10, 1.05 ± 0.11 µm2; TTX, N = 9, 0.95 ± 0.15 µm2. (C) Two additional culture averages are included that did not have corresponding mEPSC recordings. Culture #4, CON, N = 10, 0.58 ± 0.07 µm2; TTX, N = 10, 0.71 ± 0.04 µm2; Culture #5, CON, N = 10, 0.89 ± 0.05 µm2; TTX, N = 10, 0.95 ± 0.08 µm2. (D) Box plots of cell mean mEPSC amplitudes pooled from same three cultures as shown in (A). CON, N = 23 cells, 13.7 ± 0.9 pA; TTX, N = 24 cells, 16.4 ± 0.9 pA. (E) Box plots of dendrite mean GluA2 receptor cluster size pooled from same three cultures as shown in (A) and (B). CON, N = 29 dendrites, 0.90 ± 0.06 µm2; TTX, N = 28 dendrites, 1.08 ± 0.10 µm2. (F) Box plots of dendrite mean GluA2 receptor cluster size pooled from the same five cultures shown in (C). CON, N = 49 dendrites, 0.83 ± 0.04 µm2; TTX, N = 48 dendrites, 0.98 ± 0.07 µm2. Line series, Student’s paired t-test; box plots, Kruskal–Wallis test. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10 and 90 percentiles; open circles indicate means of individual cells/dendrites; bin size 0.1 pA, 0.05 µm2; line, median; dot, mean. For all p-values, * with underline indicates significance with p < 0.05.
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Figure 5—source data 1
Source data for Figure 5 comparing the TTX effect on mEPSC amplitudes and GluA2 receptors in matched and unmatched Rab3a+/+ cortical neuron cultures.
- https://cdn.elifesciences.org/articles/90261/elife-90261-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Comparison of miniature excitatory postsynaptic current (mEPSC) amplitudes and GluA1 receptor cluster areas in matched mouse cortical cultures prepared from Rab3a+/+ mice and treated with TTX for 48 hr.
(A) Culture averages of mEPSC amplitudes for untreated (CON) and TTX-treated coverslips (TTX) in each of two Rab3a+/+ mouse cortical co-cultures. Culture #2, CON, N = 7, 13.8 ± 2.4 pA; TTX, N = 8, 17.7 ± 1.8 pA (+28.3%); Culture #6, CON, N = 6, 13.0 ± 1.5 pA; TTX, N = 6, 18.5 ± 2.0 pA (+42.3%). (B) Culture averages of GluA1 receptor cluster size in the same two cultures as shown in (A). Culture #2, CON, N = 10, 0.37 ± 0.04 µm2; TTX, N = 10, 0.40 ± 0.06 µm2 (+8.1%) Culture #6, CON, N = 10, 0.35 ± 0.05 µm2; TTX, N = 10, 0.33 ± 0.04 µm2 (−5.7%) ‘Culture #2’ is the same Culture #2 depicted in Figure 5, different coverslips were processed for GluA1 immunofluorescence labeling. GluA1 receptor cluster intensity did not increase in Culture #2 (CON, 364 vs. TTX, 357) or Culture #6 (CON, 357 vs. TTX, 363). Live cultures were exposed to GluA1 antibody against the extracellular domain (ABN241, purchased from EMD Millipore, now available from Millipore Sigma) before being fixed and processed with secondary antibodies.
Figure 6
Rab3a in neurons, not astrocytes, was required for full TTX-induced homeostatic plasticity.
(A–C) Miniature excitatory postsynaptic current (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 s–1; TTX, 3.48 ± 0.64 s–1); from four 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 s–1; TTX, 4.47 ± 1.53 s–1); from two 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 s–1; TTX, 3.02 ± 0.70 s–1); from three cultures. Box plot parameters: box ends, 25th and 75th percentiles; whiskers, 10th and 90th percentiles, bin size 0.1 pA; open circles represent means from individual cells; line, median; dot, mean. p-values (shown on the graphs) are from Tukey’s post hoc test following a two-way ANOVA. p-values denoted with * and underline indicate significance of p < 0.05.
Tables
Table 1
Comparison of miniature excitatory postsynaptic current (mEPSC) amplitude and GluA2 receptor cluster characteristics in mouse cortical cultures prepared from Rab3a+/+ mice and Rab3a−/− mice.
*p < 0.05, Kruskal–Wallis test.
| Rab3a+/+ | Rab3a−/− | |||||||
|---|---|---|---|---|---|---|---|---|
| Quantity | CON, mean, SEM | TTX, mean, SEM | % change | p-value | CON, mean, SEM | TTX, mean, SEM | % change | p-value |
| mEPSC amplitude | 13.7 ± 0.9 pA (23 cells, 3 cultures) | 16.4 ± 0.9 pA (24 cells, 3 cultures) | +19.7 | *0.02 | 14.9 ± 0.8 pA (21 cells, 3 cultures) | 13.5 ± 0.9 pA (21 cells, 3 cultures) | −9.4 | 0.18 |
| GluA2 cluster size | 0.83 ± 0.04 µm2 (49 cells, 5 cultures) | 0.98 ± 0.07 µm2 (48 cells, 5 cultures) | +18.1 | 0.21 | 0.93 ± 0.05 µm2 (30 cells, 3 cultures) | 0.91 ± 0.05 µm2 (29 cells, 3 cultures) | −2.2 | 0.75 |
| GluA2 cluster intensity | 733 ± 14 a.u. (49 cells, 5 cultures) | 738 ± 13 a.u. (48 cells, 5 cultures) | +0.7 | 0.69 | 766 ± 12 a.u. (30 cells, 3 cultures) | 776 ± 15 a.u. (29 cells, 3 cultures) | +1.3 | 0.46 |
| GluA2 cluster integral | 311,021 ± 19,282 a.u. (49 cells, 5 cultures) | 365,366 ± 27,080 a.u. (48 cells, 5 cultures) | +17.4 | 0.22 | 369,436 ± 23,439 a.u. (30 cells, 3 cultures) | 364,237 ± 25,833 a.u. (29 cells, 3 cultures) | −0.8 | 0.86 |
Table 2
Comparison of miniature excitatory postsynaptic current (mEPSC) amplitude and VGLUT1-positive presynaptic site characteristics in mouse cortical cultures prepared from Rab3a+/+ and Rab3a−/− mice.
mEPSC data are identical to that in Table 1, reproduced here for comparison purposes. *p < 0.05, Kruskal–Wallis test.
| Rab3a+/+ | Rab3a−/− | |||||||
|---|---|---|---|---|---|---|---|---|
| Quantity | CON, mean, SEM | TTX, mean, SEM | % change | p-value | CON, mean, SEM | TTX, mean, SEM | % change | p-value |
| mEPSC amplitude | 13.9 ± 1.1 pA (23 cells, 3 cultures) | 16.7 ± 0.9 pA (24 cells, 3 cultures) | +20.1 | *0.01 | 14.9 ± 0.8 pA (21 cells, 3 cultures) | 13.5 ± 0.9 pA (21 cells, 3 cultures) | −9.4 | 0.18 |
| VGLUT1 site size | 1.17 ± 0.08 µm2 (29 cells, 3 cultures) | 1.07 ± 0.06 µm2 (24 cells, 3 cultures) | −8.5 | 0.97 | 0.89 ± 0.03 µm2 (30 cells, 3 cultures) | 0.95 ± 0.04 µm2 (29 cells, 3 cultures) | +6.7 | 0.55 |
| VGLUT1 site intensity | 699 ± 39 a.u. (29 cells, 3 cultures) | 639 ± 31 a.u. (28 cells, 3 cultures) | −8.6 | 0.28 | 554 ± 13 a.u. (30 cells, 3 cultures) | 570 ± 19 a.u. (29 cells, 3 cultures) | +2.9 | 0.18 |
| VGLUT1 site integral | 430,787 ± 36,818 a.u. (29 cells, 3 cultures) | 351,653 ± 21,689 a.u. (28 cells, 3 cultures) | −18.4 | 0.13 | 257,159 ± 18,265 a.u. (30 cells, 3 cultures) | 289,857 ± 18,521 a.u. (29 cells, 3 cultures) | +12.7 | 0.21 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (include species and sex here) | Rab3A knockout strain, C57BL/6J; (Mus musculus, mixed sex) | The Jackson Laboratory | B6;129S-Rab3atm1Sud/J (strain #: 002443); RRID:IMSR_JAX:002443 | |
| Strain, strain background (include species and sex here) | Rab3A earlybird strain, C57BL/6J; C3HeJ (Mus musculus, mixed sex) | Kapfhamer et al., 2002 | ||
| Genetic reagent (Escherichia coli) | Bsp1286I | New England Biolabs | Cat. #: R0120S | Restriction enzyme |
| Antibody | anti-GluA (mouse, extracellular, monoclonal: clone 6C4) | Millipore | Cat. #: MAB397 RRID:AB_2113875 | 1:40 |
| Antibody | anti-MAP2 (chicken, polyclonal) | Abcam | Cat. #: ab5392 RRID:AB_2138153 | 1:2500 |
| Antibody | anti-VGLUT1 (rabbit, polyclonal) | Synaptic Systems | Cat. #: 135 303 RRID:AB_887875 | 1:4000 |
| Sequence-based reagent | D8Mit31-F | This paper | Rab3A PCR Primers (forward) | TCC TGT GAC CTC CAA CTG TG |
| Sequence-based reagent | D8Mit31-R | This paper | Rab3A PCR Primers (reverse) | GGC CCA AAA CTG AGC AAC |
| Sequence-based reagent | RabF1 | This paper | Rab3A ebd PCR Primers (forward) | TGA CTC CTT CAC TCC AGC CT |
| Sequence-based reagent | Dcaps3R | This paper | Rab3A ebd PCR Primers (reverse) | TGC ACT GCA TTA AAT GAC TCC T |
| Chemical compound, drug | Tetrodotoxin (TTX) | Tocris | Cat. #: 1069 | |
| Chemical compound, drug | N-naphthyl acetylspermine (NASPM) | Tocris | Cat. #: 2766 | |
| Chemical compound, drug | Picrotoxin | Sigma-Aldrich | P1675 | |
| Software, algorithm | MiniAnalysis | Synaptosoft | ||
| Other | Papain | Worthington Biochemical | LK003178 | Enzyme for neuronal cell dissociation |
| Other | Neurobasal-A | Gibco | 10888022 | Neuronal cell culture media |
| Other | B27 | Gibco | 17504044 | Serum-free culture media supplement |
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Homeostatic synaptic plasticity of miniature excitatory postsynaptic currents in mouse cortical cultures requires neuronal Rab3a
eLife 12:RP90261.
https://doi.org/10.7554/eLife.90261.5
