Spontaneous neurotransmission signals through store-driven Ca2+ transients to maintain synaptic homeostasis
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Multiple approaches to detect miniature spontaneous Ca2+ transients (mSCTs) in the presence of TTX and physiological Mg2+.
(A) Loading dissociated rat hippocampal cultures with Fluo-4 AM dye labels all cells on the coverslip (above) and produces the largest signal amplitudes, shown as example traces and an average with …
Figure 2 with 1 supplement
Detection and characterization of spontaneous Ca2+ transients in physiological concentrations of Mg2+.
(A) Events detected from Fluo-4 AM traces having rising slope greater than 350 fluorescence units/s and a peak ∆F/Fo greater than 0.035 were counted if the peak fluorescence value was 2 standard …
Figure 2—figure supplement 1
Syb2-mOrange fluorescence does not contaminate Fluo-4 AM signals.
(A) Example traces from simultaneous Fluo-4 AM / synaptobrevin-mOrange imaging. Cells were imaged alternating the excitation between Fluo-4 AM and Syb2-mO for every other frame. Each frame was …
Figure 3
mSCT frequency is correlated with mEPSC frequency.
(A) Whole cell recordings from WT cells (left) show a ∼twofold increase in mEPSC frequency when switched to Tyrode's solution containing 100 mM sucrose (right) (p = 0.002, Student's paired T test, N …
Figure 4 with 2 supplements
Spontaneous Ca2+ transient generation is decreased by blocking release of Ca2+ release from internal stores but not by blocking the AMPA receptors, L-type Ca2+ channels or group I mGluRs.
(A) Traces (top) from images taken before and after treatment with the AMPA receptor blocker NBQX show no change in Ca2+ transient frequency (bottom) (p = 0.78 with Student's paired t-test, N = 9 …
Figure 4—figure supplement 1
Nimodipine produces positive results in a separate assay.
(A) Example traces showing Ca2+ influx during action potentials in the absence and presence of nimodipine. Cells were loaded with Fluo-4 AM and imaged in Tyrode's solution containing 1.25 mM Mg2+, 2 …
Figure 4—figure supplement 2
YM202074 and Fenobam produce positive results in a separate assay.
(A) Cells loaded with Fluo-4 AM were imaged in Tyrode's solution containing 1.25 mM Mg2+, 2 mM Ca2+ and TTX with or without the mGluR1/5 blockers YM202074 and fenobam. The solution was then changed …
Figure 5
Treating cells with ryanodine + TTX produces a protein synthesis dependent increase in mEPSC frequency and amplitude indicative of homeostatic synaptic scaling.
(A) Example voltage clamp recordings from cells treated with TTX + vehicle (negative control, N = 9 cells from 5 coverslips, 3 cultures), TTX + ryanodine (N = 8 cells from 5 coverslips, 4 cultures) …
Figure 6
Ryanodine treatment does not trigger homeostatic synaptic scaling in eEF2 kinase knockout neurons.
(A) Example traces from WT littermate mice with and without 3 hr ryanodine treatment. (B) Cumulative probability histogram shows a significant shift in mEPSC amplitude in TTX + ryanodine treated …
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