Parvalbumin-expressing interneurons linearly control olfactory bulb output

Abstract

In the olfactory bulb, odor representations by principal mitral cells are modulated by local inhibitory circuits. While dendrodendritic synapses between mitral and granule cells are typically thought to be a major source of this modulation, the contributions of other inhibitory neurons remain unclear. Here we demonstrate the functional properties of olfactory bulb parvalbumin-expressing interneurons (PV cells) and identify their important role in odor coding. Using paired recordings, we find that PV cells form reciprocal connections with the majority of nearby mitral cells, in contrast to the sparse connectivity between mitral and granule cells. In vivo calcium imaging in awake mice reveals that PV cells are broadly tuned to odors. Furthermore, selective PV cell inactivation enhances mitral cell responses in a linear fashion while maintaining mitral cell odor preferences. Thus, dense connections between mitral and PV cells underlie an inhibitory circuit poised to modulate the gain of olfactory bulb output.

Figure 1. Intrinsic and synaptic properties of olfactory bulb PV cells

(A) Olfactory bulb schematic. OSNs: olfactory sensory neurons, PV cells: parvalbumin-expressing cells. Each color in the OSNs represents OSNs that express a particular odorant receptor. (B) Overlay of tdTomato (red) and DAPI (blue) channels of a parasagittal section (50 μm) of olfactory bulb from a mouse derived from crossing the lines PV-Cre and Rosa-LSL-tdTomato. Glom: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer, GCL: granule cell layer. (C) Anatomical reconstructions of two representative PV cells. Lines at the top and bottom of each cell represent the borders between layers. (D) Current-clamp recording of bottom cell in (C). Responses to a series of hyperpolarizing and depolarizing current steps (100 pA increments) are shown. Strong depolarization elicits a delayed burst of high frequency spikes. Note the high frequency of spontaneous EPSPs evident in subthreshold traces. (E) Olfactory sensory nerve stimulation evokes prolonged barrages of excitatory synaptic responses. Top, PV cell in current-clamp (spike truncated), bottom, same cell in voltage-clamp (V_m = −80 mV) configuration. Inset: recording schematic. (F) Mitral cell layer stimulation evokes fast, inwardly-rectifying EPSCs with little contribution of slow NMDARs at depolarized membrane potentials. Top, recording schematic. Left, current-voltage relationship of mitral cell-evoked EPSCs in a representative PV cell. Right, average current-voltage relationship (black circles, error bars = SEM, n = 5 cells) of mitral cell-evoked EPSCs normalized to the amplitude recorded at −80 mV. Dashed line represents linear fit to the responses between −80 and −20 mV. (G) Summary plot (average and SEM, n = 5 cells) showing that philanthotoxin-433 (PhTx, 10 μM), a selective blocker of GluA2-lacking AMPARs, strongly reduces the amplitude of mitral cell-evoked EPSCs in PV cells. The remaining EPSC was completely blocked by subsequent application of the AMPA receptor antagonist NBQX (10 μM). Inset: responses from a representative cell under control conditions, 20 min following application of PhTx, and subsequent application of NBQX. See also Figure S1.

Figure 2. Dense reciprocal connectivity between mitral and PV cells

(A₁) Simultaneous recording of a synaptically-connected mitral cell-PV cell pair. Left: an action potential in a mitral cell evokes an EPSC in a PV cell. Right: in the same pair of cells, an action potential in the PV cell evokes an IPSC in the mitral cell. Top: recording schematic. (A₂) Simultaneous recording of a synaptically-connected mitral cell-granule cell pair. Mitral cell action potential evokes an EPSC in the granule cell. (A₃) Summary of connection probabilities indicating that connectivity of mitral cells onto PV cells is substantially higher than that onto granule cells. Almost all (82%) connections between mitral and PV cells are reciprocal (solid red bar). (B) Trains of action potentials (20 Hz) in connected mitral-PV cell pairs elicit depressing synaptic responses. Top: reciprocally connected cell pair showing that both mitral cell synapses onto PV cells (left) and PV cell synapses onto mitral cells (right) depress during stimulus trains. Bottom: summary plot (MC to PV: black circles, n = 29 pairs and PV to MC: open circles, n = 9 pairs) showing average response amplitude normalized to the first action potential of the trains. Error bars represent SEM. (C) and (D) Light activation of ChR2-expressing PV cells drives inhibition onto mitral cells but not granule or other PV cells. (C) Left: responses of simultaneously recorded mitral and granule cells to PV cell photoactivation. Blue ticks represent LED illumination. Right: summary data of light-evoked IPSC amplitudes in all cell pairs (n = 7). (D) Left: responses of simultaneously recorded mitral and PV cells to photoactivation. Right: summary data of light-evoked IPSC amplitudes in all cell pairs (n = 6).

Figure 3. Broad odor tuning of PV cells revealed by in vivo calcium imaging

(A) In vivo imaging configuration. (B) In vivo two-photon images of GCaMP5G-expressing PV cells, granule cells, and mitral cells (three separate mice). (C) Individual PV cells are activated by a broad range of odors, while mitral and granule cell responses are more odor selective. Top: responses of three simultaneously imaged PV cells to a panel of seven odors (see Experimental Procedures for the identity of odors). Middle: responses of three granule cells to the same set of odors. Bottom: responses of three mitral cells to the same set of odors. Gray: individual trials; black: averaged trace. (D) Summary cumulative fraction distributions of odor tuning broadness (the number of odors eliciting responses out of the seven tested odors) showing that PV cells are far more broadly tuned to odors than mitral or granule cells. Black: mitral cells (n = 7 mice, 290 cells). Blue: granule cells (n = 3 mice, 375 cells). Red: PV cells (n = 5 mice, 69 cells). (E) Top: odor-evoked activity maps of PV cells from one animal in response to three different odors (pseudocoloring represents odor-evoked GCaMP5G dF/F response averaged across seven trials). Each odor activates virtually all PV cells in the imaging field. Middle: odor-evoked activity maps of granule cells. Bottom: odor-evoked activity maps of mitral cells. For both granule and mitral cells, each odor activates overlapping but distinct subpopulations of cells. Left panels show all imaged cells in white (ROIs). (F) Correlation matrices depicting the pairwise similarity between population activity patterns evoked by seven different odors. Top: matrix created from PV cell activity. Middle: matrix created from granule cells. Bottom: matrix created from mitral cells. The correlation coefficients are higher in PV cells compared to granule or mitral cells. PV cells: n = 69 cells; mitral cells: n = 290 cells; granule cells: n = 375 cells. See also Figure S2 and S3.

Figure 4. PV cell activity is strongly enhanced by increases in respiration rate

(A) Top: in vivo imaging configuration with mouse on a circular treadmill. Bottom: example traces of simultaneously recorded respiration and running speed. Spontaneous running is accompanied by an increase in respiration frequency. (B) Running-related increases in respiration rate are associated with uniform increases in PV cell activity, while mitral and granule cell activity is variably modulated by changes in respiration. (B₁) Top: respiration rate during a 55 sec imaging session. Bottom, simultaneously imaged activity of ten PV cells. Cells are sorted in descending order based on correlation coefficient values between the cell activity and respiration frequency. All PV cells show increases in fluorescence during periods of high frequency respiration. (B₂) Granule cells (n = 64 cells) and (B₃) mitral cells (n = 80 cells) show more variable responses to changes in respiration frequency. (C) Normalized fluorescence intensities of individual cells shown in (B) binned with respect to respiration frequency. Fluorescence intensities are normalized to values when respiration frequency was 1–3 Hz. (C₁) Activity in PV cells increases linearly with elevations in respiration frequency. Red line: average. Gray lines: individual cells. (C₂)Mi tral and (C₃) granule cell activity show both increases and decreases in fluorescence with elevations in respiration rate. (C₄) Plugging the ipsilateral nostril blocks the respiration-modulation of PV cell activity. Error bars represent SEM. (D) Summary data of the correlations between respiration frequency and normalized fluorescence intensity (slopes of the regression lines) shown as box plots where whiskers represent most extreme values within 1.5 × I.Q.R. and outliers shown in red crosses. (PV cells: 4 mice, 38 cells; granule cells: 4 mice, 329 cells; mitral cells: 5 mice, 201 cells; PV cells with plug: 3 mice, 36 cells).

Figure 5. Pharmacogenetic suppression of PV cells in vivo

(A) Schematic illustrating the expression of GCaMP5G and PSAM^L141F-GlyR (PSAM) in PV cells. The PSAM agonist PSEM³⁰⁸ (PSEM) is injected intraperitoneally to suppress PV cell activity. (B)Co -expression of PSAM and GCaMP5G in PV cells imaged in vivo. Left: GCaMP5G fluorescence. Middle: dTomNLS fluorescence indicating PSAM expression. Right: merged image showing the co-localization of GCaMP5G (green) and PSAM (red) in PV cells. (C) Odor-evoked responses of the same PV cell before and after PSEM injection. Odor responses are transiently blocked following PSEM injection at Time 0 min and gradually recover to baseline levels over ~40 min. (D) Activity maps of PV cell population responses show that odor-evoked responses are strongly reduced following PSEM injection. Before: average across three trials before PSEM injection. PSEM: average across three trials following PSEM injection (8–24 min postinjection). Recovery: average across three trials one hour after PSEM injection. Left panel shows all imaged cells in white. (E) Change index (CI, see Experimental Procedures) summary of PV cell population activity for each trial. PSEM injection rapidly suppresses odor responses of PV cells in PSAM-expressing mice (filled circles, n = 3 mice, 35 cells), while having no effect on responses from non-PSAM-expressing control mice (open circles, n = 3 mice, 31 cells). Error bars represent SEM. See also Figure S4.

Figure 6. Suppression of PV cells linearly enhances mitral cell odor-evoked activity

(A) Schematic illustrating mitral cell imaging, with PSAM expressed specifically in PV cells. The PSAM agonist PSEM is injected intraperitoneally to suppress PV cells. (B) Mitral cells and PV cells imaged sequentially from a representative mouse. Left: mitral cells expressing GCaMP5G. Right: PV cells in EPL expressing PSAM and dTomNLS. (C) Activity maps of mitral cell odor-evoked responses before and after PSEM injection. Top: a mouse expressing GCaMP5G in mitral cells and PSAM in PV cells. Mitral cell ensembles respond more strongly to the same odor after PSEM injection. Bottom: same conditions as above, but in a control mouse without viral expression of PSAM in PV cells. PSEM injection has no obvious effect on mitral cell population activity. Left panels, mitral cell ROIs from each animal. (D) Top, odor-evoked responses of a representative mitral cell before and after PSEM injection. Gray dotted line represents the peak response amplitude before PSEM injection. Bottom, change index of mitral cell odor-evoked responses for each trial. PSEM injection transiently increases odor responses of mitral cells in PSAM-expressing mice (filled circles, n = 4 mice, 146 cells), while having no effect on responses from non-expressing control mice (open circles, n = 4 mice, 147 cells). Error bars represent SEM. (E) PV cell suppression enhances mitral cell activity in a multiplicative manner without altering odor preferences. (E₁) Tuning curves of four representative cells from PSAM-expressing mice. Odors were ranked for each cell according to the response amplitude during baseline trials. Black: three trials before PSEM injection. Red: three trials immediately after PSEM injection. (E₂) Tuning curves averaged across all cells from PSAM-expressing mice that showed responses to at least three out of seven tested odors (n = 67 cells, 4 mice). Blue dotted line represents a curve based on linear transformation (1.30 × original response + 0.09). The equation was determined using the maximum and minimum values of the experimental data. (F) Peak response amplitudes under control conditions (‘baseline’, before PV cell inactivation) plotted against response amplitudes during PV cell inactivation for all cell-odor pairs which were judged as responsive during control conditions (n = 366 cell-odor pairs). Linear regression fit (red; not forced to the origin) yields a slope greater than one and intercept close to zero, indicating a linear enhancement of mitral cell responses during PV cell inactivation. Gray dotted line: unity. (G) Relationship between mitral cell population activity (population dF/F) before and during PV cell inactivation (n = 28 mouse-odor pairs) indicates a linear transformation (linear fit with slope >1 and intercept near zero) of population activity during PV cell inactivation. Red line: linear regression, gray dotted line: unity.