Balancing Extrasynaptic Excitation and Synaptic Inhibition within Olfactory Bulb Glomeruli

Spillover-mediated currents are due to glutamate release from eTCs. A, Recording from an eTC-MC pair showing MC currents (blue; at V_hold = –77 mV) evoked by single eTC spikes (black; in LCA mode). Raw traces (left) and averages (n = 94) are shown. Note the amplitude and kinetic similarities to MC currents recorded in the PG cell-MC pairs (Fig. 1B4). B, Comparison of the magnitude (B1) and kinetic properties (B2; 20%-to-80% rise-time and half-width) of MC currents recorded in eTC-MC pairs (n = 9) versus PG cell-MC pairs (n = 8 for B1, n = 7 for B2). Lines in B1 reflect mean ± SEM. Integrated charge values were multiplied by –1. C, Current events collected from a triple-cell recording that included a same-glomerulus eTC, PG cell, and MC. In the expanded and normalized average traces (boxed inset), it is clear that the PG cell and MC currents both had 1- to 2-ms onset delays after the eTC spike, indicating that the eTC was the source of the currents.

Ultrastructural evidence for complexes that could support spillover. A, Electron micrographs (A1, A2; two images are four 50-nm sections apart) and three-dimensional reconstruction (A3) of an example complex that includes a DAB-labeled eTC dendrite (darkened in micrographs; light yellow in reconstruction) forming a synapse (green) onto a putative PG cell dendrite (blue). A glutamatergic dendrite that was assigned to be a putative MC (red; see main text) and glial processes (purple) are in close proximity. Note that the glial processes in the reconstruction appear to surround the dendrites. Inset in A1, Same image as in A1 but without the colors so that the synapse (green arrow) can be seen more clearly. Scale bars: 0.5 μm for micrographs, 0.1 μm for inset of A1. Scale cube in A3 = 0.5 µm³. B, Another example of a complex containing an eTC-to-PG cell synapse with adjacent glial process and putative MC dendrite. Note that in the reconstruction the putative MC dendrite runs behind the PG cell and eTC dendrites. Scale bars and cube as in A. C, Electron micrograph (C1) and reconstruction (C2) of a cluster of synapses (in red) from OSNs (green) onto a DAB-labeled eTC dendrite (light yellow in reconstruction). Note the absence of an adjacent putative MC dendrite. Inset in C1, Original image of the eTC dendrite showing one of the OSN synapses (red arrow) that is indicated by the white dashed box. Scale bars and cube as in A. D, Summary of analysis of 13 eTC-to-PG cell synapses and 39 OSN-to-eTC synapses (pooled results from analysis of two eTC fills in two bulb slices). The fraction out of the total with an adjacent putative MC dendrite (within 0.5 µm) was much higher for eTC-to-PG cell synapses.

Absence of spillover at glutamatergic axonal synapses in glomeruli. A, Model to be tested: glutamate released at axonal synapses onto eTC dendrites spills over and activates glutamate receptors on adjacent MC dendrites. B, Example whole-cell recording from an eTC-MC pair at the same glomerulus used to test for spillover at axonal synapses. Raw traces recorded without a stimulus (B1), an image of the pair (B2; collapsed in the z-axis), and examples and averages (n = 293) of detected sEPSCs in the eTC and time-locked MC currents (B3) are illustrated. The cell body of the eTC in this pair was just above the glomerulus to which the MC sent its apical dendrite. C, Plot relating the magnitude of the sEPSCs and associated MC currents for the experiment in B. Line reflects linear regression fit of the data (r = –0.012, p = 0.82). D, Summary of MC currents recorded in eTC-MC pairs (open squares) versus PG cell-MC pairs (filled circles), plotted as a function of the amplitude of the fast EPSC in the reference cell (either eTC or PG cell). Data were binned according to the magnitude of the fast EPSCs in 100-pA/ms increments. Note that, while the MC current in the PG cell-MC pairs clearly increased as a function of the magnitude of the PG cell EPSC (linear regression fit: r = 0.79, p = 5.6 × 10⁻⁵), no such relationship was observed in the eTC-MC pairs (r = 0.28, p = 0.31). The eTC-MC pair dataset includes four to five points each in the bins centered at 50, 150, and 250 pA. Each point in each bin reflects a single experiment; *p = 0.01 in Mann–Whitney U test comparing the MC current in the two cell-pair types. E, Electrophysiological evidence that the eTC and MC in B were affiliated with the same glomerulus. E1, Two response trials overlaid showing that OSN stimulation (38 µA; at arrow) evoked LLDs in both cells in trial 2, but not trial 1. E2, Summary plot of responses to OSN stimulation for the same experiment (49 trials) relating the magnitude of the delayed current in the two cells. Note the clustering of data points in the upper right, corresponding to cooccurring LLD events. Current magnitude measurements were obtained by integrating the current starting 50 ms after OSN stimulation to avoid the OSN-EPSC. Line reflects linear regression fit (r = 0.98, p < 1.0 × 10⁻⁶).

Supralinear increase in extrasynaptic excitatory currents. A, Example whole-cell recordings from an eTC-MC pair illustrating MC current responses to different numbers of action potentials (APs) in the eTC. eTC spikes were evoked by direct depolarizing current pulses (500 pA, 25 ms). Note in the raw traces (A1; three superimposed trials) and averages (A2) that the total current associated with I_extra for more than or equal to two eTC spikes was much larger than linear summation of the current driven by one AP. In one of the trials in which the eTC spiked seven times in the same experiment (A3), the MC response included a distinct, much larger LLD event ∼150 ms after the onset of I_extra (note scale bar). B, Another eTC-MC pair recording in which the number of spikes in the eTC recorded in the LCA mode (B1; raw example trace) was related to I_extra in the MC (B2; averages). C, Summary of I_extra measurements from 12 pair recordings versus number of spikes in the eTC (N). Values, which reflect experiments in which the eTCs were in whole-cell (open squares) or LCA (open circles) patch modes, were normalized to the charge elicited by one eTC spike in the same pair. Note that nearly all points lie above the dashed line, which reflects linear summation of I_extra elicited by one eTC spike. Most pair recordings contributed multiple data points to the plot. D, Supralinearity indices S_N measured across 12 pair-cell recordings separated by recording type for the eTC. A single value is plotted for each pair-cell recording, reflecting the average I_extra measured whenever the eTC fired at least two spikes (N ≥ 2). Asterisks: p ≤ 0.041 in comparison to S_N≥2 = 1 (linear), paired t tests. Lines reflect mean ± SEM for each recording type. E1, Supralinearity indices S_N (mean ± SEM) sorted by the number of spikes in the eTC. Each data point reflects 8–10 recordings obtained when the eTC was in either whole-cell or LCA patch mode. Superimposed line reflects fit of the individual S_N values across all experiments, which yielded a significant correlation coefficient (0.37, p = 0.031). E2, Similar to part E1, except that values for the supralinearity indices were adjusted (yielding S_N’) for the relative amount of glutamate released across different numbers of eTC spikes. Glutamate release was estimated from EPSCs recorded in eTC-PG cell pairs (Fig. 6); x-axis values reflect estimates of the amount of glutamate release following two, three, four, or five to seven eTC spikes, normalized to the single spike-evoked release in the same recording.

Sublinear increase in PG cell excitatory currents in eTC-PG cell pairs. A, Example recordings in an eTC-PG cell pair showing that spontaneous bursts of spikes in the eTC (recorded in LCA mode) were associated with strongly depressing rapid EPSCs in the PG cell. Three selected examples with varying number of eTC spikes are illustrated in A1. Analysis of 37 such bursts in this pair indicated that the degree of synaptic depression as reflected in the amplitude ratio of the first two EPSCs in the burst (Amp₂/Amp₁) was negatively correlated with the first EPSC amplitude (r = –0.63, p < 0.0001; A2) and positively correlated with the interval between the first two EPSCs (r = 0.44, p = 0.0058; A3). Both are consistent with a presynaptic vesicle depletion mechanism for depression. B, Summary of integrated current measurements from 10 eTC-PG cell pair recordings, demonstrating that the synaptic depression resulted in a sublinear increase in the excitatory current as a function of spike number in the eTC. Diagonal line reflects linearity. C, Example PG cell recording showing that the AMPA receptor allosteric modulator CTZ (100 µM) prolonged the EPSCs without altering the degree of depression. Selected EPSC bursts under each condition are illustrated in C1, while EPSC half-widths (C2) and amplitude ratios (Amp₂/Amp₁; C3) for the same experiment are also shown. The absence of an effect of CTZ on depression supported a presynaptic mechanism. D, Histograms summarizing CTZ effects on EPSC half-width (top) and the Amp₂/Amp₁ ratio (bottom) from five PG cell recordings; *p = 0.020 in paired t test. E, Estimates of the magnitude of slow currents in PG cells not directly associated with rapid EPSCs. PG cell currents, both under control conditions and in CTZ, were measured in a 100- to 200-ms window after the start of each eTC spike burst for bursts with more than or equal to four spikes lasting <100 ms. MC currents (reflecting I_extra) measured in the same manner are also plotted; n = 5 for each recording type; *p < 0.01 in paired t test comparison with zero current. F, Summary of absolute charge measurements as a function of eTC spike number for I_extra in MCs in the eTC-MC pairs (blue) and the PG cell current in the eTC-PG cell pairs (red). Each data point reflects mean ± SEM from 7–10 recordings; *p < 0.01 in Mann–Whitney U test, Bonferroni correction for multiple comparisons.

Tests of different mechanisms that could underlie the supralinear increase in extrasynaptic excitatory currents. A, Example eTC-MC pair recordings showing effects of the glial glutamate transporter blocker DL-TBOA (50 µM) on I_extra. Note that DL-TBOA enhanced I_extra when the eTC fired five spikes (left; averages of 12–15 bursts), but not when the eTC spiked only once (right; averages of 60). An increase that is selective for the multi-spike condition is inconsistent with glutamate transporter saturation being the cause of the supralinear increase in I_extra (see main text). B, Summary of the effect of DL-TBOA on I_extra separated by when the eTC spiked once (n = 5 pairs) versus two or more times (n = 6); *p = 0.0012 in unpaired t test comparing more than or equal to two- and one-spike datasets. Lines reflect mean ± SEM for the two conditions. C, Example recording of I_extra in MCs (averages of 12–15 trials) induced by eTC spike bursts (five spikes/burst) showing that the current was mostly blocked by the AMPA and NMDA receptor blockers, NBQX (10 µM) plus DL-AP5 (50 µM). This argued against the supralinear increase in I_extra being due to recruitment of a neurotransmitter other than glutamate. D, Effect of NQBX plus DL-AP5 as a function of the magnitude of I_extra elicited by more than or equal to two eTC spikes. Superimposed fitted line indicates lack of correlation (p = 0.25), providing further evidence against the largest I_extra signals being due to a non-glutamate neurotransmitter. Each of the eight data points reflects one eTC-MC pair recording. E, Test for recurrent excitation and asynchronous glutamate release. E1, Example analysis of a single burst event in an eTC-PG cell pair in the no-LLD condition. Note that each eTC spike had a single time-locked EPSC reflecting monosynaptic transmission, also evident in the derivative trace at bottom (horizontal dashed line = threshold). The absence of excess events argued against recurrent excitation or asynchronous glutamate release being the cause of the supralinear increase in I_extra. Box demarcates a burst event in the same recording in which there was a cooccurring LLD; in this example we counted 12 excess EPSCs in the PG cell. E2, Histogram reflecting latencies between eTC spikes and PG cell EPSCs in the no LLD-condition for the experiment in E1. Latencies of 0.5–3.5 ms (open bars) were considered to reflect monosynaptic transmission from the test eTC onto the PG cell. F, Distribution of the number of excess PG cell EPSCs per burst across five eTC-PG cell pair recordings under the no LLD-condition. Note that the large majority of burst events had no excess PG cell EPSCs.