Calcium-Independent Inhibitory G-Protein Signaling Induces Persistent Presynaptic Muting of Hippocampal Synapses

Depolarization-induced presynaptic silencing is not dependent on increases in intracellular free calcium from any source. A, Representative examples of sucrose-evoked EPSCs and action potential-evoked EPSCs elicited in autaptic neurons after 4 h treatment in normal calcium-containing media (control) or calcium-free media supplemented with 20 μm of the cell-permeant fast calcium chelator BAPTA-AM. B, Summary of results for sucrose-evoked EPSC charge and action-potential-evoked EPSC amplitude after treatment with control or with calcium-free, BAPTA-AM-supplemented extracellular media in sister cultures (n = 9–11 neurons). *p < 0.05. C, Example fields of neurons filled with the calcium indicator fluo-4 before (0 mm) and after (0.5 mm) perfusion of CaCl₂. Neurons were pretreated for 4 h with 30 mm KCl in either calcium-containing media (depolarized) or calcium-free media supplemented with 20 μm BAPTA-AM (depolarized BAPTA-AM). Scale bar, 50 μm. D, Representative examples of sucrose-evoked EPSCs in autaptic neurons after 4 h treatment in calcium-free media supplemented with 20 μm BAPTA-AM and either 30 mm NaCl (baseline) or 30 mm KCl (depolarized).

Activation of inhibitory G-proteins is necessary for depolarization-induced presynaptic silencing. A, Example action-potential-evoked EPSCs from autaptic neurons treated 24 h with or without 500 ng/ml pertussis toxin before 4 h coapplication with 30 mm NaCl (baseline) or 30 mm KCl (depolarized). B, Summary of EPSC amplitudes from autaptic neurons treated as in A (n = 13–14 neurons). *p < 0.05 with Bonferroni's correction for multiple comparisons. C, Example images of vGluT-1 (red) and FM1-43FX (green) correspondence in neurons treated 24 h with or without 500 ng/ml pertussis toxin before 4 h NaCl (baseline) or KCI (depolarized). Scale bar, 5 μm. D, Summary of conditions represented in C (n = 25 fields from 5 independent experiments). *p < 0.05 with Bonferroni's correction for multiple comparisons.

A₁ adenosine receptor activation is not necessary for depolarization-induced presynaptic silencing. A, Example action-potential-evoked EPSCs from autaptic neurons treated 4 h with 30 mm NaCl (baseline) or 30 mm KCl (depolarized) in the presence or absence of 200 nm DPCPX, an A₁ receptor antagonist. B, Summary of EPSC amplitudes from autaptic neurons treated as in A (n = 9 neurons). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.). C, Example images of vGluT-1 (red) and FM1-43FX (green) correspondence in neurons treated 4 h under baseline or depolarized conditions in the presence or absence of 200 nm DPCPX. Scale bar, 5 μm. D, Summary of conditions represented in C (n = 65 fields from 13 independent experiments). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.).

None of likely candidate GPCRs is necessary for depolarization-induced silencing. A, Example action-potential-evoked IPSCs from a single autaptic neuron after acutely applied extracellular saline (control), 10 μm baclofen (a GABA_B receptor agonist), or 10 μm baclofen plus 50 μm SCH50911 (a GABA_B receptor antagonist). B, Summary of experiments measuring vGluT-1/FM1-43FX correspondence in neurons treated 4 h with 30 mm NaCl (baseline) or 30 mm KCl (depolarized) in the presence or absence of 50 μm SCH50911 (n = 35 fields from 7 independent experiments). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except the one indicated (N.S.). C, Example action-potential-evoked EPSCs from a single autaptic neuron after acutely applied control, 50 μm ACPD (an mGluR agonist), or 50 μm ACPD plus 25 μm LY341495 (a mGluR antagonist). D, Summary of experiments measuring vGluT-1/FM1-43FX correspondence in neurons treated for 4 h under baseline or depolarized conditions in the presence or absence of 25 μm LY341495 (n = 25 fields from 5 independent experiments). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.). E, Example action potential-evoked EPSCs from two different autaptic neurons: one after acutely applied control or 200 nm WIN55,212–2 (a CB₁ agonist) and the other after 1–2 h pretreatment with 1 μm AM251 (a CB₁ antagonist) and continued AM251 application or AM251 plus WIN55,212-2. The AM251 pretreatment with between-cell comparison was used to mitigate against the very slow reversibility of WIN55,212-2. F, Summary of experiments measuring vGluT-1/FM1-43FX correspondence in neurons treated for 4 h under baseline or depolarized conditions in the presence or absence of 1 μm AM251 (n = 20 fields from 4 independent experiments). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.). G, Summary of experiments measuring vGluT-1/FM1-43FX correspondence in neurons treated for 4 h under baseline or depolarized conditions in the presence or absence of 200 nm DPCPX, 50 μm SCH50911, 25 μm LY341495, and 1 μm AM251 (cocktail; n = 20 fields from 4 independent experiments). All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.). H, Summary of experiments measuring EPSC amplitude in neurons treated 4 h under baseline or depolarized conditions in the presence of either 1 μm NBQX or 10 μm NBQX to ensure kainate receptor block (n = 14–16 neurons). Neurons treated for 4 h with 1 μm NBQX were treated acutely (∼1 min) with 10 μm NBQX before recording as a control for any residual acute NBQX effects. All comparisons were significantly different (p < 0.05 with Bonferroni's correction for multiple comparisons) except those indicated (N.S.).

A₁ adenosine receptor activation and GABA_B receptor activation induce presynaptic silencing. A, Example images of vGluT-1 (red) and FM1-43FX (green) correspondence in neurons treated 4 h with control or 100 μm adenosine in the presence or absence of 200 nm DPCPX. Scale bar, 5 μm. B, Summary of conditions represented in A (n = 40 fields from 8 independent experiments). *p < 0.05 with Bonferroni's correction for multiple comparisons. C, Summary of experiments measuring vGluT-1/FM1-43FX correspondence in neurons treated 4 h with control medium, with 10 nm CCPA, or with 10 nm CCPA coapplied with 200 nm DPCPX (n = 35 fields from 7 independent experiments). *p < 0.05 with Bonferroni's correction for multiple comparisons. D, Example action-potential-evoked EPSCs recorded from autaptic neurons after acute (<1 min; control) or 4 h treatment with 10 nm CCPA. E, Summary of EPSC amplitudes from autaptic neurons treated acutely (<1 min; control) or for 4 h with 50 μm baclofen (n = 9–10 neurons) and FM1-43FX/vGluT-1 correspondence after 4 h control or 50 μm baclofen treatment (n = 30 fields from 6 independent experiments). *p < 0.05.