Fig 1.
TRPC channels modulate the vesicle replenishment rate and the RRP size.
(A) Exemplary EPSCs triggered by HFS (20 Hz, 40 AP/2 s) of autaptic wt and TRPC1/C4/C5 tko neurons (inset magnified view on the first 4 EPSCs of the train response. Arrows depict the synchronous and asynchronous phase of secretion). (B and C) TRPC deficiency reduces the EPSC amplitudes and accelerates the time course of synaptic depression (In panel C, data were normalized to the first EPSC amplitude). (D) The first EPSC amplitude is significantly reduced in tko cells (wt, n = 63; tko, n = 39 cells; p = 1.9 × 10−9). (E and F) The AmpEPSC10/AmpEPSC1 ratios of wt neurons range from STD to STE. Loss of TRPCs shifts the AmpEPSC10/AmpEPSC1 ratio towards STD. (G) The AmpEPSC10/AmpEPSC1 ratio is reduced in tko cells (p = 0.00067). (H, I, and J) The total synchronous and asynchronous charges are smaller in tko cells (red) when compared with controls (black). (K) Quantification of the 40th asynchronous charge (p = 0.0000056). (L) Mean cumulative synchronous release components during the 20-Hz train. Continuous line, linear regression of the last 5 data points back-extrapolated to stimulus = 0 to estimate the initial RRP size. Note that the RRP analysis was confined to wt neurons that reached a steady-state response in the late phase of stimulus train (45 out of 63 neurons). (M) The RRP size is significantly decreased in tko cells (wt, n = 45; tko, n = 39; p = 0.0000013). N. The release probability remained unchanged between groups (p = 0.2). (O) Cumulative total charge during 20-Hz train stimulation. The slope of the linear regression from the last 4 stimulation points (continuous line) rendered an estimate of the replenishment rate in panel P. (P) The replenishment rate is significantly reduced in tko neurons (p = 1.6 × 10−10). **p < 0.01; ***p < 0.001; statistical significance was assessed by Mann-Whitney rank sum test. Underlying data can be found in S1 Data. Amp, amplitude; AP, action potential; EPSC, evoked postsynaptic current; HFS, high-frequency stimulation; RRP, readily releasable pool; STD, short-term depression; STE, short-term enhancement; tko, triple knockout; TRPC, transient receptor potential canonical; wt, wild type.
Fig 2.
Endogenous TRPC5 channels promote STE of synaptic signaling.
(A) Exemplary epifluorescence image of an autaptic TRPC5-IC/eR26-τGFP neuron. (B) Sample recordings from tko, τGFP-negative and τGFP-positive neurons upon 20-Hz HFS. (C–E) τGFP-positive cells show a strong STE of synaptic signaling and an increased PPR (panel E, EPSC2/EPSC1) when compared with tko or τGFP-negative neurons (τGFP-positive versus tko p = 0.00005; τGFP-positive versus τGFP-negative p = 0.003). (F and G) Frequency and cumulative frequency distribution of the AmpEPSC10/AmpEPSC1 ratios for tko (red), τGFP-negative (black), and τGFP-positive neurons (green). (H) The AmpEPSC10/AmpEPSC1 ratio is higher in τGFP-negative cells than in tko neurons and even further increased in τGFP-positive cells (τGFP positive versus tko p = 7.892264 × 10−9; τGFP positive versus τGFP negative p = 0.0009; τGFP negative and tko p = 0.02). (I) Time course of total charge during HFS. (J and K) The replenishment rate determined from the cumulative charge plot shown in panel J gradually increases between tko, τGFP-negative, and τGFP-positive neurons (τGFP positive versus tko p = 1.5 × 10−10; τGFP positive versus τGFP-negative p = 0.0001; τGFP negative and tko p = 0.002). (L and M) Time courses of synchronous and asynchronous charge transfer during the stimulus train. (N and O) The 10th EPSC charge and the 40th asynchronous charge are significantly increased in τGFP-negative and -positive neurons (10th charge: τGFP positive versus tko p = 1.7 × 10−8; τGFP positive versus τGFP negative p = 0.003; τGFP negative and tko p = 0.002; 40th charge: τGFP positive versus tko p = 0.00004; τGFP poisitive versus τGFP negative p = 0.004; τGFP negative and tko p = 0.018). Data were collected from tko (n = 25), τGFP-negative (n = 38), and τGFP-positive (n = 40) neurons. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA on ranks followed by Dunn’s post hoc test; significance was also tested by Mann-Whitney rank sum test for τGFP-negative cells versus tko. Underlying data can be found in S1 Data. Amp, amplitude; AP, action potential; EPSC, evoked postsynaptic current; eR26, ROSA26-floxed-stop; HFS, high-frequency stimulation; IC, internal ribosomal entry site cre recombinase; PPR, paired-pulse ratio; STD, short-term depression; STE, short-term enhancement; tko, triple knockout; TRPC, transient receptor potential canonical; τGFP, τ-green fluorescent protein.
Fig 3.
Expression of either TRPC1 or TRPC5 in wt neurons turns synaptic depression into STE.
(A) Sample recordings of EPSCs triggered by HFS (20 Hz, 40 AP/2 s) of autaptic wt neurons and those expressing TRPC5 or TRPC1. (B) Activity-dependent increase in the EPSC amplitudes in wt cells expressing C1 or C5. (C) TRPC expression causes STE of synaptic signaling (data were normalized to the initial peak EPSC amplitude). (D and E) Expression of TRPC1 or TRPC5 shifts the frequency distribution of the AmpEPSC10/AmpEPSC1 ratios to higher values. (F) Mean AmpEPSC10/AmpEPSC1 ratio for the indicated groups (+C1, p = 0.002; +C5, p = 0.0001; versus wt). (G) Mean paired pulse ratio (+C1, p = 0.0036; +C5, p = 0.00585; versus wt). (H) Time course of total charge transfer. (I) Mean cumulative total charge transfer during HFS. The replenishment rate was determined from the slope of the cumulative plot (last 4 data points). (J) The replenishment rate is significantly enhanced in cells expressing either C1 or C5 (+C1, p = 0.003; +C5, p = 0.00001; versus wt). (K and L) Time courses of synchronous (K) and asynchronous charge transfer (L) during the stimulus train. (M and N) The 10th EPSC charge (M) and the 40th asynchronous charge (N) are significantly increased with TRPC expression (10th charge: +C1, p = 0.00001; +C5, p = 0.0000001; versus wt; 40th asynchronous: +C1, p = 0.02; +C5, p = 0.0005; versus wt). Data were collected from the following number of cells: wt, n = 43; wt + TRPC1, n = 30; wt + TRPC5, n = 24; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA on ranks followed by Dunn’s post hoc test. Underlying data can be found in S1 Data. Amp, amplitude; AP, action potential; EPSC, evoked postsynaptic current; HFS, high-frequency stimulation; PPR, paired-pulse ratio; STD, short-term depression; STE, short-term enhancement; TRPC, transient receptor potential canonical; wt, wild type.
Fig 4.
Buffering presynaptic [Ca]i abolishes TRPC-dependent short-term facilitation.
(A) Representative EPSC traces for wt neurons and those expressing TRPC1 or TRPC5 after EGTA-AM treatment. EGTA abolishes asynchronous secretion causing a phase of steady-state synchronous secretion and prevents the TRPC-mediated STE of synaptic signaling. (B). Neither TRPC expression (C1 or C5) nor pretreatment with EGTA affects the first EPSC amplitude of the train response (between groups without EGTA: p = 0.7; in-group with versus without EGTA: wt, p = 0.48; +C1, p = 0.49; +C5, p = 0.67). (C) EGTA significantly reduces the PPR for all groups (in-group with versus without EGTA: wt, p = 0.03; +C1, p = 0.001; +C5, p = 2 × 10−6). (D, F, H) Activity-dependent changes of EPSC amplitude (D), synchronous release (F), and asynchronous release (H) for wt neurons and those expressing TRPC1 or TRPC5 without (left panels) and with EGTA treatment (right panels). (E and G) The TRPC-mediated increase in the AmpEPSC10/AmpEPSC1 ratio (E) and the 40th synchronous EPSC charge (panel G, last pulse of the train shown in panel F) are prevented with EGTA treatment (EPSC10/EPSC1 [between groups without EGTA]: +C1, p = 0.07; +C5, p = 0.05, versus wt; in-group with versus without EGTA: wt, p = 0.5; +C1, p = 0.00004; +C5, p = 0.028; 40th synchronous [between groups without EGTA]: +C5, p = 0.002, +C1, p = 0.0014 versus wt; in-group with versus without EGTA: wt, p = 0.36; +C1, p = 0.0004; +C5, p = 5.8 × 10−9). (H and I) EGTA treatment diminishes asynchronous secretion in all groups, indicating effective buffering of presynaptic [Ca]i (+C5, p = 0.0008, +C1, p = 0.05; in-group with versus without EGTA: wt, p = 1.4 × 10−12; +C1, p = 0.0000003; +C5, p = 5.8 × 10−9). Data was collected from EGTA-treated wt (n = 45), wt + TRPC1 (n = 21), and wt + TRPC5 (n = 21) cells and nontreated wt (n = 13), wt + TRPC1 (n = 9), and wt + TRPC5 (n = 13) cells. *p < 0.05, one-way ANOVA on ranks followed by Dunn’s post hoc test for groups without EGTA; **p < 0.01, ***p < 0.001, Mann-Whitney U rank sum test for the in-group comparison with and without EGTA treatment. Underlying data can be found in S1 Data. EGTA-AM, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; EPSC, evoked postsynaptic current; ns, not significant; PPR, paired-pulse ratio; STE, short-term enhancement; TRPC, transient receptor potential canonical; wt, wild type.
Fig 5.
Homomeric TRPC5 channels promote strong STE of synaptic signaling in tko neurons.
(A) Sample recordings of EPSCs triggered by HFS (20 Hz, 40 AP/2 s) of tko neurons and those expressing C1 or C5. (B) Starting from similar initial EPSC amplitudes, only TRPC5 expression causes STE of the synaptic response. (C) EPSC amplitude changes during HFS normalized to the amplitude of the first response. (D–F) The AmpEPSC10/AmpEPSC1 ratio of TRPC5-expressing tko neurons is shifted to the STE range and nearly 3-fold higher compared with tko and tko + C1 responses (panel F, +C1, p = 0.14; +C5, p = 1.9 × 10−10, versus tko). (G–I) TRPC5, but not TRPC1, expression elevates the total synaptic charge transfer and increases the replenishment rate (+C1 p = 0.1; +C5, p = 2 × 10−10, versus tko). (J and K) Time courses of synchronous (J) and asynchronous charge transfer (K) during the stimulus train. (L) The 40th asynchronous charge is significantly increased with TRPC5 expression (+C1, p = 0.98; +C5, p = 0.000005; versus tko). Data were collected from the following number of neurons: tko, n = 31; tko + TRPC1, n = 26; tko + TRPC5, n = 20; **p < 0.01, ***p < 0.001; one-way ANOVA on ranks followed by Dunn’s post hoc test. Underlying data can be found in S1 Data. Amp, amplitude; AP, action potential; EPSC, evoked postsynaptic current; HFS, high-frequency stimulation; ns, not significant; STD, short-term depression; STE, short-term enhancement; tko, triple knockout; TRPC, transient receptor potential canonical.
Fig 6.
Englerin A activates presynaptic TRPC channels and increases the mEPSC frequency.
(A) Exemplary recordings of autaptic wt and TRPC1/C4/C5-tko neurons superperfused with Ringer (R) solution containing Englerin A (Eng, 1 μM). Englerin A evoked an inward current in wt but not tko cells. Right panels, expanded timescale of the recording at the indicated time points. Note the clear mEPSC frequency increase in wt neurons with Englerin A application (2). (B) Percentage of cells responding to Englerin A with an inward current (p = 0.008). (C) Quantification of the maximum inward current amplitude (wt, n = 36; tko, n = 16; p = 4.3 × 10−9) determined at the end of Englerin A application relative to baseline current. (D) Time course of the averaged inward current (black) and the corresponding mEPSC frequency (green) during Englerin A application (40–70 s) in wt neurons (n = 11). (E) mEPSC amplitudes (determined for the cells shown in panel D) remain unchanged during Englerin A application. Insets depict averaged mEPSCs during Ringer (R, n = 72) and Englerin A (E, n = 65) application. (F) Englerin A evokes a 2-fold increase in mEPSC frequency (relative to the mEPSC frequency before drug application) in wt but not in tko neurons (wt, n = 36; tko, n = 16; p = 0.00004). **p < 0.01; ***p < 0.001, Mann-Whitney rank sum test. Underlying data can be found in S1 Data. mEPSC, miniature excitatory evoked postsynaptic current; tko, triple knockout; TRPC, transient receptor potential canonical; wt, wild type.
Fig 7.
Elevated Ca2+ entry through VGCCs increases the EPSC amplitude and accelerates synaptic depression.
(A) Representative EPSC recordings of a wt neuron (20 Hz, 40 AP/2 s) before and after TEA application (300 μM). (B) Time course of the EPSC amplitude for the first (Ringer) and the second train response (+TEA); right panel, data normalized to the first EPSC amplitude. Note that TEA increases the degree of STD. (C and D) TEA increases the initial EPSC amplitude (C) and decreases the PPR (D) (first amp: p = 0.00003; PPR: p = 0.0002). (E) Time course of the EPSC synchronous charge for Ringer and TEA (left) and its cumulative plot (right). Continuous line, linear regression of the last 5 data points to estimate the initial RRP size (shown in panel F). (F and G) The RRP size (F) and the Pr (G) are significantly larger with TEA (RRP: p = 0.0007; Pr: p = 0.24). (H) TEA increases asynchronous release. (I) Mean asynchronous release of the 40th EPSC (p = 0.0001). (J) Time course of the cumulative total synaptic charge transfer; dashed lines, linear regression of the last 4 data points to estimate the replenishment rate shown in panel K. (K) TEA elevates the replenishment rate (p = 0.003). Data was collected from 16 cells, *p < 0.05; **p < 0.01; ***p < 0.001; Student paired t test. Underlying data can be found in S1 Data. AP, action potential; EPSC, evoked postsynaptic current; PPR, paired-pulse ratio; Pr, release probability; RRP, readily releasable pool; STD, short-term depression; TEA, tetraethylammonium; VGCC, voltage-gated calcium channel; wt, wild type.
Fig 8.
TRPC channels augment the presynaptic Ca2+-rise upon HFS.
(A) Exemplary images of neurons expressing the vesicular protein SybII-mRFP (left) and Synaptophysin-GCaMP6s (SyGCaMP6s, middle). Note the high degree of colocalization between synaptobrevinII and SyGCaMP6s (right). (B) Sample difference images (ΔF/F0) of SyGCamp6s signaling recorded (5 Hz) from autaptic neurons (wt, left; tko, middle; wt + TRPC5, right) during the HFS (20 Hz, 2 s); insets are corresponding EPSC recordings from the same cell. Arrows are exemplary ROIs used to monitor ΔF/F0 at single synaptic sites. (C) Loss of TRPC channels decreases the presynaptic Ca2+ rise, whereas expression of either TRPC variant strongly increases the Ca2+ signal. (D) Maximum ΔF/F0 from the data shown in (C), tko, p = 0.006; +C1, p = 0.0076; +C5, p = 0.0023. (E) TRPC deficiency decreases, whereas TRPC expression increases the slope of ΔF/F0 during HFS (slope between the 6th and the 13th data point; tko, p = 0.003; +C1, p = 0.035; +C5, p = 0.002. (F) Expansion of the early phase of the plot shown in panel C illustrating the prolonged Ca2+ rise after HFS. (G) Expression of either TRPC variant significantly enhances the Ca2+ influx right after HFS (slope determined between the 14th and 20th data point; tko, p = 0.48; +C1, p = 0.025; +C5, p = 0.0003). (H) Corresponding mean cumulative total charge transfer of the neurons imaged in panel C. (I and J) The replenishment rate (determined from the slope of the cumulative plot, last 4 data points, shown in panel H, is significantly changed by altering TRPC expression (I) and correlates with changes in SyGCaMP6s (slope of ΔF/F0) during HFS); tko, p = 0.0002; +C1, p = 0.009; +C5, p = 0.0004. Data were collected from wt, n = 15; tko, n = 9; wt + TRPC1, n = 11; wt + TRPC5, n = 15; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA on ranks followed by Dunn’s post hoc test versus wt. wt versus tko Mann-Whitney rank sum test. Underlying data can be found in S1 Data. AP, action potential; EMCCD, electron multiplying charge coupled device; EPSC, evoked postsynaptic current; HFS, high-frequency stimulation; mRFP, monomeric red fluorescent protein; ROI, region of interest; SybII, synaptobrevin II; SyGCaMP6s, Synaptophysin-GCaMP6s; tko, triple knockout; TRPC, transient receptor potential canonical; VM, membrane voltage; wt, wild type; ΔF, delta fluorescence.