Intrinsic Conductances Actively Shape Excitatory and Inhibitory Postsynaptic Responses in Olfactory Bulb External Tufted Cells

Persistent Na⁺ conductance amplifies subthreshold postsynaptic excitatory responses and produces temporal summation. A, Typical recording showing that EPSC is completely blocked by NBQX (10 μm). B, Inverted NBQX-sensitive EPSC from A. C, Comparison of two _simEPSPs evoked by injecting the inverted EPSC in B from the same cell with holding potential at either −55 (black) or −75 mV (green). D, Pooled data from five cells showing that amplitude (left) and decay time constant (right) of _simEPSP at −55 mV is significantly greater than at −75 mV (n = 5). E, Comparison of two _simEPSPs from the same cell at −55 mV before (black) and after (red) TTX (1 μm). F, Plots showing both amplitude (left) and decay (right) of _simEPSP from five cells at −55 mV are reduced by TTX (1 μm; n = 5). G, Comparison of responses to injection of a train of five inverted EPSCs at 40 Hz before (black) and after (red) TTX (1 μm). H, Plots showing that TTX significantly reduces the peak amplitude (left; n = 5) of the second to the fifth _simEPSPs and the integrated area (right; n = 5) under five _simEPSPs evoked by the train of inverted EPSCs. Error bars indicate SEM. *p < 0.05; **p < 0.01.

Voltage dependence of suprathreshold and subthreshold postsynaptic responses. A, B, Current-clamp recordings showing the effect of holding membrane potential (from −53 to −69 mV; 2 mV increments) on suprathreshold postsynaptic firing responses to ON stimulation (A) and subthreshold _simEPSPs evoked by an inverted EPSC (20 pA) injection in the absence (black) or presence (red) of 1 μm TTX (B). C, Plot of six cells showing that ON-evoked spikes per response significantly increase with membrane depolarization in the range from −59 to −53 mV. D, E, Plots showing that both amplitude (D) and half-width (E) of _simEPSPs from five cells are significantly reduced by TTX (1 μm) in the voltage range from −57 to −53 mV. *p < 0.05; **p < 0.01. Error bars indicate SEM.

Low-voltage-activated Ca²⁺ conductance voltage-dependently boosts subthreshold synaptic responses. A, Current-clamp recording showing that ON-evoked EPSP is boosted by Bay K8644 (Bay K; 5 μm, 5 min) in the presence of 10 nm TTX. Replacing Bay K8644 with both NNC55-0396 (NNC; 50 μm) and nimodipine (20 μm) for 10 min not only reverses the amplificatory effect but further attenuates EPSP compared with control. B, Plots showing that amplitude (left) and half-width (right) of ON-evoked EPSPs from five cells are significantly and reversibly enhanced by Bay K8644 (5 μm, 5 min) with holding potential at −65 mV. Note both amplitude and half-width of EPSPs after the replacement of Bay K8644 with both NNC55-0396 (50 μm) and nimodipine (20 μm) for 10 min are significantly smaller than in control. C, Current-clamp recordings showing that large but not small _simEPSPs are enhanced by Bay K8644 (5 μm, 5 min). D, Pooled data from five cells showing that both the amplitude (left) and half-width (right) of large (top) but not small (bottom) _simEPSPs are enhanced by Bay K8644 (5 μm, 5 min) with the holding potential at −55 mV. E, Current-clamp recording showing that large but not small _simEPSPs are attenuated by nimodipine (20 μm, 10 min). F, Graphs showing that both the amplitude (left) and half-width (right) of large (top) but not small (bottom) _simEPSPs from five cells are reduced by NNC55-0396 (50 μm, 10 min) with the holding potential at −55 mV. G, Recording showing that large but not small _simEPSPs are attenuated by NNC55-0396 (50 μm, 10 min). Nim., Nimodipine. H, Plots showing that both amplitude (left) and half-width (right) of large (top) but not small (bottom) _simEPSPs from five cells are reduced by nimodipine (20 μm, 10 min) with the holding potential at −55 mV. Error bars indicate SEM. **p < 0.01; ***p < 0.001.

T-type Ca²⁺ conductance amplifies suprathreshold synaptic responses. A, Current-clamp recording of suprathreshold postsynaptic responses to ON-stimulation before (left) and after (right) NNC55-0396 (50 μm, 10 min) treatment with holding potential at −60 mV. B, Pooled data from five cells showing that NNC55-0396 turns burst-firing (right) into single-spike-firing (left) suprathreshold responses. C, Plot showing the effect of sequentially bath-applied gabazine (10 μm), CGP55845 (10 μm), and l-741626 (5 μm) on the spikes per suprathreshold response. D, Pooled data from five cells showing that bath application of mixed blockers (10 μm gabazine, 10 μm CGP55845, and 5 μm l-741626) for 10 min significantly increases spikes per suprathreshold synaptic response; addition of NNC55-0396 (NNC; 50 μm, 10 min) in the presence of the blocker mixture still reduces spikes per suprathreshold synaptic response in the same cells. Bar graphs in D represent average value. ***p < 0.001.

Blocking L-type Ca²⁺ conductance attenuates suprathreshold synaptic responses. A–C, Current-clamp recordings showing postsynaptic responses to suprathreshold ON-stimulation in the same cell before (A), after nimodipine (20 μm) treatment for 10 min (B), and after replacing nimodipine with Bay K8644 (5 μm) for 10 min (C). D–F, Pooled data from five cells showing the spikes per suprathreshold response before (D), after nimodipine treatment for 10 min (E), and after washout of nimodipine with addition of Bay K8644 for 10 min (F). G, Plot showing the increment of spikes per response in five cells by mixture of synaptic blockers (10 μm gabazine, 10 μm CGP55845, and 5 μm l-741626) for 10 min. Nimodipine (nim.; 20 μm, 10 min) significantly decreases spikes per suprathreshold response in the presence of blocker mixture in the same cells. Bar graphs represent the average values. H, Recording of paired-pulse EPSCs with interpulse interval at 50 ms before (left) and after NNC55-0396 (NNC; 50 μm; top right) or nimodipine (20 μm; bottom right) treatment for 10 min (holding potential, −60 mV). ***p < 0.001.

Activation of L-type Ca²⁺ conductance boosts suprathreshold postsynaptic responses. A–C, Current-clamp recordings of suprathreshold synaptic response before (A, black), after Bay K8644 (Bay K; 5 μm) for 5 min (B, red), and after replacement of Bay K8644 by nimodipine (Nim; 20 μm) for 15 min (C, green). D–F, Pooled data from five cells showing spikes per suprathreshold response before (D, black), after Bay K8644 treatment for 10 min (E, red), and after replacing Bay K8644 with nimodipine for 15 min (F, green). G, Graph showing that exposure to the mixture of synaptic blockers (10 μm gabazine, 10 μm CGP55845, and 5 μm l-741626 for 10 min) increases spikes per suprathreshold response; addition of Bay K8644 (5 μm, 5 min) further increases spikes per response. H, Plot showing that Bay K8644 significantly reduces the PPR in all five cells tested; reduced PPR is reversed by washout of Bay K8644 with the addition of nimodipine for 15 min in all three cells tested. Inset, Voltage-clamp recordings of paired EPSCs (interpulse interval 50 ms) before (black) and after (red) Bay K8644 (5 μm, 5 min). Note that the amplitude of the first EPSC from the paired-pulse EPSCs in the inset is not affected by Bay K8644 (5 μm, 10 min). I, Paired-pulse EPSCs before (black) and after (red) Bay K8644 (5 μm, 10 min) in the presence of both CGP55845 (10 μm) and l-741626 (5 μm). J, Comparison of the AHP after suprathreshold responses before (black) and after (red) Bay K8644 (5 μm, 5 min) in the absence of gabazine. K, Comparison of the AHP after suprathreshold responses in the same cell before (black), after gabazine (5 μm, 10 min; cyan), and after Bay K8644 (5 μm, 5 min; red) in the presence of gabazine. L, Graphs showing that the average AHP area integral is significantly reduced by gabazine but not affected by Bay K8644 in the presence of gabazine (n = 5 cells). **p < 0.01; ***p < 0.001. N.S., Not significant.

Blocking I_h prolongs EPSPs and produces temporal summation. A, Comparison of _simEPSPs from the same cell with holding potential at −55 or −75 mV in the presence of TTX (1 μm). Inset, The decay time constant of _simEPSPs from five cells at −55 mV is significantly higher than that from the same cells at −75 mV. B, Comparison of ON-evoked EPSPs from the same cell before (black) and after ZD7288 (10 μm, 10 min; red). C, Comparison of _simEPSPs from the same cell held at −55 mV before (black) and after ZD7288 (10 μm, 10 min; red). D, Graphs showing that the area integral (right) but not the amplitude (left) of single _simEPSPs from five cells is increased by ZD7288 (ZD). E, Trains of five _simEPSPs (40 Hz) from the same cell at −55 mV before (black) and after (red) ZD7288 (10 μm, 10 min). F, Graphs showing both the relative amplitudes (normalized to the first, left) of and the integrated area (right) under multiple _simEPSPs from five cells are significantly increased by ZD7288. *p < 0.05; **p < 0.01. N.S., Not significant.

Effects of blocking I_h on suprathreshold synaptic responses. A, B, Current-clamp recordings of suprathreshold postsynaptic response to ON stimulation before (A, black), after ZD7288 (ZD; 10 μm, 10 min; B, red), and after ZD7288 plus maintained 30 pA depolarizing current injection (B, green). C, Graph showing that the spike number per suprathreshold response from five cells is significantly reduced by ZD7288 alone, but is increased by ZD7288 with compensation of membrane potential change. D, Paired-pulse EPSCs from the same cell holding at −60 mV before (black) and after ZD7288 (10 μm, 10 min; red). Error bars indicate SEM. *p < 0.05; ***p < 0.001.