The initial synapse in the olfactory system is from olfactory nerve (ON) terminals to postsynaptic targets in olfactory bulb glomeruli. Recent studies have disclosed multiple presynaptic factors that regulate this important linkage, but less is known about the contribution of postsynaptic intrinsic conductances to integration at these synapses. The present study demonstrates voltage-dependent amplification of EPSPs in external tufted (ET) cells in response to monosynaptic (ON) inputs. This amplification is mainly exerted by persistent Na(+) conductance. Larger EPSPs, which bring the membrane potential to a relatively depolarized level, are further boosted by the low-voltage-activated Ca(2+) conductance. In contrast, the hyperpolarization-activated nonselective cation conductance (I(h)) attenuates EPSPs mainly by reducing EPSP duration; this also reduces temporal summation of multiple EPSPs. Regulation of EPSPs by these subthreshold, voltage-dependent conductances can enhance both the signal-to-noise ratio and the temporal summation of multiple synaptic inputs and thus help ET cells differentiate high- and low-frequency synaptic inputs. I(h) can also transform inhibitory inputs to postsynaptic excitation. When the ET cell membrane potential is relatively depolarized, as during a burst of action potentials, IPSPs produce classic inhibition. However, near resting membrane potentials where I(h) is engaged, IPSPs produce rebound bursts of action potentials. ET cells excite GABAergic PG cells. Thus, the transformation of inhibitory inputs to postsynaptic excitation in ET cells may enhance intraglomerular inhibition of mitral/tufted cells, the main output neurons in the olfactory bulb, and hence shape signaling to olfactory cortex.
A: Two dimensional reconstruction of a typical biocytin-filled ET cell (EPL: external plexiform layer; GL: glomerular layer; ONL: olfactory nerve layer). B: Typical voltage clamp recording (holding potential −60 mV) showing ON-evoked and AMPA/kainate receptor-mediated EPSCs with constant and short latency. C: Current clamp recording of the same cell showing ON-evoked subthreshold EPSPs and suprathreshold burst firing responses which were blocked by NBQX (10 μM). D: Plotting EPSP amplitude against stimulus intensity shows the step-like input-output relation of subthreshold responses from a typical cell. Inset: Current clamp recording of EPSPs in response to ON stimuli with incremental intensities corresponding to colored solid points. E: Input-output relation from the same cell in voltage clamp. F: Bar graph showing number of cells with number of EPSP steps evoked by incremental stimulus intensities (n=19). G: Input-output relation of suprathreshold responses from 4 cells showing that spike number per response does not continue to increase with incremental intensity of ON stimulation. H: Latency of the first spike within each burst firing response decreases with incremental ON stimulation intensity. I: Plot of the number of spikes per response to ON stimulation with intensity of 100 μA remains very stable over 20 min.
Fig. 2. Persistent Na + conductance amplifies…
Fig. 2. 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 simulated EPSPs (simEPSPs) evoked by injecting the inverted EPSC in B from the same cell with holding potential at either −55 mV (black) or −75 mV (green). D: Pooled data from 5 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 5 cells at −55 mV are reduced by TTX (1 μM, n=5). G: Comparison of responses to injection of a train of 5 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 simEPSP and the integrated area (right, n=5) under 5 simEPSPs evoked by the train of inverted EPSCs. *p<0.05; **p<0.01; ***p<0.001.
Fig. 3. Voltage dependence of supra- and…
Fig. 3. Voltage dependence of supra- and subthreshold postsynaptic responses
A & B: Current clamp…
Fig. 3. Voltage dependence of supra- 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 (A) suprathreshold postsynaptic firing responses to ON stimulation and (B) subthreshold simEPSPs evoked by an inverted EPSC (20 pA) injection in the absence (black) or presence (red) of 1 μM TTX. C: Plot of 6 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 5 cells are significantly reduced by TTX (1 μM) in the voltage range from −57 to −53 mV. *p<0.05; **p<0.01.
Fig. 4. Low voltage activated Ca 2+ …
Fig. 4. Low voltage activated Ca 2+ conductance voltage-dependently boosts subthreshold synaptic responses
A: Current clamp recording showing that ON-evoked EPSP is boosted by Bay K8644 (5 μM, 5 min) in the presence of 10 nM TTX. Replacing Bay K8644 with both NNC55-0396 (50 μM) and nimodipine (20 μM) for 10 min not only reverses the amplificatory effect but further attenuates EPSP compared to control. B: Plots showing that amplitude (left) and half-width (right) of ON-evoked EPSPs from 5 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 5 cells showing that both amplitude (left) and half-width (right) of large (top) but not small (bottom) simEPSPs are enhanced by Bay K8644 (5 μM, 5 min) with 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 amplitude (left) and half-width (right of large (top) but not small (bottom) simEPSPs from 5 cells are reduced by nimodipine (20 μM, 10 min) with holding potential at −55 mV. G: Recording showing that large but not small simEPSPs are attenuated by NNC55-0396 (50 μM, 10 min). H: Plots showing that both amplitude (left) and half-width (right) of large (top) but not small (bottom) simEPSPs from 5 cells are reduced by NNC55-0396 (50 μM, 10 min) with holding potential at −55 mV. **p<0.01; ***p<0.001.
Fig. 5. T-type Ca 2+ conductance amplifies…
Fig. 5. T-type Ca 2+ 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 5 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 5 cells showing that bath application of cocktailed 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 (50 μM, 10 min) in the presence of the blocker cocktail still reduces spikes per suprathreshold synaptic response in the same cells. Bar graphs represent average value. ***p<0.001.
A, B & C: Current clamp recordings showing postsynaptic responses to suprathreshold ON-stimulation in the same cell before (A), after (B) nimodipine (20 μM) treatment for 10 min and after replacing nimodipine with Bay K8644 (5 μM) for 10 min (C). D, E & F: Pooled data from 5 cells showing the spikes per suprathreshold response before (D), after (E) nimodipine treatment for 10 min and after washout of nimodipine with addition of Bay K8644 for 10 min (F). G: Plot showing increment of spikes per response in 5 cells by cocktail of synaptic blockers (10 μM gabazine, 10 μM CGP55845 and 5 μM L-741626) for 10 min. Nimodipine (20 μM, 10 min) significantly decreases spikes per suprathreshold response in the presence of blocker cocktail in the same cells. Bar graphs represent the average values. H: Recording of paired-pulse EPSCs with inter-pulse interval at 50 ms before (left) and after NNC55-0396 (50 μM, right top) or nimodipine (20 μM, right bottom) treatment for 10 min (holding potential -60 mV). I: ***p<0.001.
Fig. 7. Activation of L-type Ca 2+ …
Fig. 7. Activation of L-type Ca 2+ conductance boosts suprathreshold postsynaptic responses
A, B & C: Current clamp recordings of suprathreshold synaptic response before (A, black), after (B, red) Bay K8644 (5 μM) for 5 min and after replacement of Bay K8644 by nimodipine (20 μM) for 15 min (C, green). D, E & F: Pooled data from 5 cells showing spikes per suprathreshold response before (D, black), after (E, red) Bay K8644 treatment for 10 min and after replacing Bay K8644 with nimodipine for 15 min (F, green). G: Graph showing that exposure to the cocktail 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 Bay K8644 significantly reduces paired-pulse ratio (PPR) in all 5 cells tested; reduced PPR is reversed by washout Bay K8644 with addition of nimodipine for 15 min in all 3 cells tested. Inset: Voltage clamp recordings of paired EPSCs (inter-pulse interval 50 ms) before (black) and after (red) Bay K8644 (5 μM; 5 min). Note 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 afterhyperpolarization (AHP) following suprathreshold responses before (black) and after (red) Bay K8644 (5 μM, 5 min) in the absence of gabazine. K: Comparison of the AHP following suprathreshold responses in the same cell before (black), after (cyan) gabazine (5 μM, 10 min) and after Bay K8644 (5 μM, 5 min, red) in the presence of gabazine. L: Graphs showing 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.
Fig. 8. Blocking I h prolongs EPSPs…
Fig. 8. Blocking I h prolongs EPSPs and produces temporal summation
A: Comparison of sim EPSPs…
Fig. 8. Blocking Ih prolongs EPSPs and produces temporal summation
A: Comparison of simEPSPs from the same cell with holding potential at -55 mV or -75 mV in the presence of TTX (1 μM). Inset showing the decay time constant of simEPSPs from 5 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 (red) ZD7288 (10 μM, 10 min). C: Comparison of simEPSPs from the same cell holding at -55 mV before (black) and after (red) ZD7288 (10 μM, 10 min). D: Graphs showing that the area integral (right) but not the amplitude (left) of single simEPSPs from 5 cells is increased by ZD7288. E: Trains of 5 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 5 cells are significantly increased by ZD7288. *p<0.05; **p<0.01.
Fig. 9. Effects of blocking I h …
Fig. 9. Effects of blocking I h on suprathreshold synaptic responses
A, B: Current clamp recordings…
Fig. 9. Effects of blocking Ih on suprathreshold synaptic responses
A, B: Current clamp recordings of suprathreshold postsynaptic response to ON stimulation before (A, black), after (B, red) ZD7288 (10 μM, 10 min) and after ZD7288 plus maintained 30 pA depolarizing current injection (B, green). C: Graph showing the spike number per suprathreshold response from 5 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 (red) ZD7288 (10 μM, 10 min). *p<0.05; ***p<0.001.
Fig. 10. I h transforms inhibitory inputs…
Fig. 10. I h transforms inhibitory inputs to postsynaptic excitation
A: Voltage clamp recordings of ON-evoked…
Fig. 10. Ih transforms inhibitory inputs to postsynaptic excitation
A: Voltage clamp recordings of ON-evoked EPSCs from the same cell with holding potential at either −20 mV or 0 mV before (black) and after (red) gabazine (10 μM, 10 min) showing GABAA receptor-mediated IPSCs. Note that the IPSC onset latency is longer than that of EPSC. B: Voltage clamp recordings of spontaneous synaptic currents from the same cell with holding potential at −55 mV, 0 mV or 0 mV in the presence of gabazine (10 μM, 10 min) showing inward spontaneous EPSCs and GABAA receptor-mediated outward spontaneous IPSCs (sIPSCs). C: Upper panel: superimposed sIPSCs from B with holding potential at 0 mV; red trace represents the average IPSC. Lower panel: an inverted average IPSC from the upper panel (left) and two inverted IPSCs paired at 50 Hz (right) are used as simulated IPSC(s) (simIPSC) to evoke voltage responses. D: Current clamp recordings showing that a rebound burst response (black) evoked by injection of the two simIPSCs in C to an ET cell at −55 mV is eliminated by ZD7288 (10 μM, red). Note that restoring hyperpolarized membrane potential caused by ZD7288 to control level does not restore the rebound burst firing response (green). E: Comparison of responses to two simIPSCs injected in the same cell held at −65 mV before (black) and after (red) ZD7288 (10 μM, 10 min) showing that the rebound depolarization is completely abolished by ZD7288. Inset: Graph showing that the rebound depolarization evoked by a train of two simIPSCs from 5 cells is completely eliminated by ZD7288. ***p<0.001.
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