Independent control of gamma and theta activity by distinct interneuron networks in the olfactory bulb

Abstract

Circuits in the brain possess the ability to orchestrate activities on different timescales, but the manner in which distinct circuits interact to sculpt diverse rhythms remains unresolved. The olfactory bulb is a classic example of a place in which slow theta and fast gamma rhythms coexist. Furthermore, inhibitory interneurons that are generally implicated in rhythm generation are segregated into distinct layers, neatly separating local and global motifs. We combined intracellular recordings in vivo with circuit-specific optogenetic interference to examine the contribution of inhibition to rhythmic activity in the mouse olfactory bulb. We found that the two inhibitory circuits controlled rhythms on distinct timescales: local, glomerular networks coordinated theta activity, regulating baseline and odor-evoked inhibition, whereas granule cells orchestrated gamma synchrony and spike timing. Notably, granule cells did not contribute to baseline rhythms or sniff-coupled odor-evoked inhibition. Thus, activities on theta and gamma timescales are controlled by separate, dissociable inhibitory networks in the olfactory bulb.

Figure 1. Efficiency and specificity of optogenetic silencing of granule cells

Assessment of specificity and effectiveness of infection in OB slices and in vivo. (a) Expression pattern in the OB following GCL injection in a Gad2-Cre animal (top). The region marked by the white box is shown zoomed in the inset. GCL, MCL and GL correspond to granule cell layer, mitral cell layer and glomerular layer. Scale bars for the overview and inset = 0.5 mm and 0.1 mm. See also the Supplementary Movie of a z-stack across the entire OB showing GFP expression pattern in the whole OB. Bottom: Immunohistochemical assessment of infection efficiency in a Vgat-Cre animal: top panel shows cre-immunoreactivity in red, middle panel shows raw GFP fluorescence in green. Bottom panel is the overlay of red and green signals. Scale bar = 50 μm. Image section taken from the center of the GCL. (b-f) Electrophysiological assessment of infection and silencing efficiency and selectivity. (b) Example whole cell recordings from MCs in OB slices from Gad2-Cre animals injected with AAV-FLEX-ArchT in the GCL during light presentation (left; yellow bar indicates light presentation). Scale bars = 20 mV, 0.5 s. Summary histogram (right) shows proportion of recorded cells with corresponding light evoked Vm deviation. Vm deviation more negative than 10 mV is marked green (“silenced”). The number on the top right corner is the total number of cells recorded. (c,d) as in (b) but recordings were from randomly selected GCs from GCL-injected Gad2-Cre (c) or Vgat-Cre (d) animals. Scale bars = 20 mV, 0.5 s. (e,f) in vivo assessment of GC silencing in Gad2-Cre, anesthetized animals (e) and Vgat-Cre, awake, head fixed animals (f). Data were from 5 and 3 animals, respectively. Traces in the middle are examples from different cells and numbers below indicate measured Vm deviation with the same colour coding as the histograms. Scale bars = 20 mV, 0.5 s. (g-i) GC silencing during excitatory odor responses in vivo. (g) Example of a reconstructed GC morphology. (h) Action potentials evoked by 1s odor presentation (gray) under control condition (top trace). Response to the same odorant during light presentation (bottom trace). Scale bar = 10 mV. (i) Summary data showing average number of APs evoked during 1s odor presentation, for odor only and with additional light presentation. n = 3 cells from 3 animals.

Figure 2. Glomerular layer inhibition structures the baseline theta rhythm

Layer selective optogenetic silencing of GABAergic neurons: AAV-FLEX-ArchT was injected into the GCL coordinates in the centre (a) or the GL coordinates superficially (b) of the OB. Green = GFP fluorescence and blue = DAPI staining. Scale bar = 0.4 mm. Inset illustrates the experimental configuration showing whole cell patch recordings made in vivo in anaesthetized Gad2-Cre or Vgat-Cre mice. (c,e) The effect of GC layer silencing on M/TC sniff-coupling; (c) Sniff-aligned, raw (top) Vm traces during control (black) and during LED presentation (green) from an example cell; nasal flow template is shown below (gray). Scale bars = 10 mV & 417.5 ms. (below) Sniff aligned, normalized average traces (from 165 sniff cycles for control and 60 cycles for LED conditions). Vertical bars above indicate the preferred phases for control (black) and LED (green) conditions. (e) Summary of phase shifts (ϕ_LED-ϕ_Cont) during GCL silencing as a polar histogram (green histogram). Baseline phase drift (ϕ_Cont1-ϕ_Cont2) is shown as dotted lines. n = 18 cells from 14 animals. (d,f) Effect of GL silencing on M/TC sniff-coupling. (d) as (c), but for GL silencing. Scale bars = 10 mV & 352.5 ms (top). Average traces were from 77 cycles (control) and 24 cycles (LED). (f) Summary phase shift histogram for GL silencing (n = 23 cells from 16 animals). (g) Dependence of GL silencing-induced phase shift on the baseline preferred phase of the recorded M/TCs (n = 23 cells, same as in f). Color indicates the likelihood of the recorded cell being TCs (cyan) or MCs (pink) based on the preferred phase of sniff coupling (see Methods; ref).

Figure 3. Sniff-coupling of distinct JGC populations

(a-d) Method used to distinguish putative PGo from other JGCs (JGr) in vivo. (a) Rationale for the method: (left) JGCs involved in the feedforward pathway (PGo) are predominantly driven by the olfactory nerve, while other types of JGCs (JGr) are significantly driven by M/TCs. (right) Activation of GCs via ChR2 suppresses M/TC activities, which in turn deprives excitatory drives to JGr cells, but not to PGo cells. (b) Example of an infected GC excited by ChR2 activation in vivo (blue bar = light presentation). (c) Example of hyperpolarization evoked in a M/TC during light presentation. (d) Summary of average M/TC firing rate for control and light-on period. n = 8 cells from 5 animals. (e) Example whole cell recordings from two JGCs showing reduction in synaptic inputs during light presentation. Such cells are classified as JGr cells. (f) Examples of cells not showing reduction in synaptic inputs, classified as PGo cells. (g) Summary histogram of changes in Vm variance during light presentation. Cells with statistically significant changes in Vm variance are classified JGr cells (gray) while those without significant change are categorized as putative PGos. (h-k) Respiratory coupling properties of PGos. (h) Example Vm trace (black) from a PGo cell, aligned to respiratory rhythm. Nasal flow template is shown below (gray). Scale bar = 10 mV, 338 ms. (i) Histogram of AP phase from all PGo cells (n=6 cells from 6 animals), normalized by the number of sniff cycles analyzed. 284 ± 86 cycles per cell analyzed. Sniff-aligned average subthreshold Vm from morphologically identified MCs (n = 7 MCs; data reproduced from ref ) is overlaid for comparison (pink). Data is repeated over 2 cycles for illustration. (j) Sniff-aligned subthreshold Vm from all PGos (mean and 1 standard deviation shown). Pink trace is the same as in (i). (k) Summary polar histogram of preferred Vm phase of all PGos (gray), normalized by the total number of cells. Pink range indicates the preferred phase of MC hyperpolarization (ticks = 25, 50 & 75 percentiles, corresponding to 1.5, 2.0 & 3.0 radians). Scale bar = π/6 radians corresponding to 32.1 ms when calibrated for the average cycle lengths of all cells analyzed. (l-o) Respiratory coupling properties of JGr cells, as above. 412 ± 139 cycles per cell analysed. n = 10 JGr cells from 8 animals. Scale bar in (l) is 10 mV, 403 ms. Scale bar in (o) corresponds to 32.7 ms.

Figure 4. GL inhibition, rather than GC lateral inhibition, underlies slow odor-evoked inhibition

Odors presented to the animal often evoke sniff-coupled hyperpolarization in M/TCs as shown by two examples (a). Ticks represent the times of expirations peaks. Scale bars = 10 mV, 0.5 s. (b) For all cell-odor pairs (n = 205 pairs, 55 cells), 36% showed detectable responses, of which 21% are purely hyperpolarizing. (c) Two hypotheses for the source of the evoked inhibition: (left) M/TCs that receive excitatory input from neighbouring glomeruli activate GCs, which in turn give lateral inhibition to the recorded cell. (right) Feedforward inhibition within the glomerulus underlies evoked inhibition. (d,e) AAV-FLEX-ArchT was injected into the GCL and glomerular layer of Gad2-Cre or Vgat-Cre animals for layer-selective silencing as in Fig. 3. (d) Effect of GC silencing: Example Vm traces from a M/TC (top) during control (black) and during light presentation (green). Odor presentation (gray) was for 1 s and overlapped with light presentation (yellow) for the LED condition. Scale bars = 10 mV, 0.5 s. (below) Summary of evoked Vm during control (odor only) vs. during GC silencing (+LED). Mean ± s.e.m. shown. n = 19 cells from 13 animals. Odors used = isoamylacetate, methylsalicylate and eugenol at 2-5% saturated vapor. (e) Effect of GL silencing: example traces & summary as in (e). n = 7 cells from 5 animals.

Figure 5. Feedforward circuit in the GL likely mediates the evoked inhibition

Pharmacological block of phasic GABA_A activation (GABA_A-clamp; ref ) in vivo, in anesthetized C57/Bl6 mice by superfusion of gabazine (0.45 mM) and muscimol (2 mM) for at least 30 minutes. (a) Example traces from an M/T cell that showed odor evoked inhibition during control (black) and the effect of GABA_A-clamp (green) on this response. Dotted lines indicate the average Vm during baseline period. Scale bars = 10 mV, 0.5 s. (bottom) Summary of evoked potential during control and GABA_A-clamp conditions where odor evoked inhibition during control. Mean ± s.e.m. shown. n = 11 cell-odor pairs from 8 animals. (b) as in (a) but for cell-odor pairs where odor did not evoke detectable response during control. n = 7 cell-odor pairs from 3 animals. Odors used = isoamylacetate, eugenol, cinnamaldehyde, salicylaldelhyde, annisaldehyde. See also Supplementary Fig. 4.

Figure 6. Granule cells coordinate neuronal activities on a fast timescale

(a) Spectral density estimate for odor period normalized by that for baseline period (“evoked power”) averaged over all recordings (n = 10 recordings from 10 animals). Black = odor only, green = odor + LED. Mean and one standard deviation shown. Example spectrograms with odor only (top) and with additional GCL silencing (yellow bar; bottom). Hotter colors indicate greater power. Spectrograms are the average from 17 trials each. (c) Summary of evoked power averaged over gamma frequencies (40-100 Hz). Mean ± s.e.m. shown. (d-f) Gamma range activities on a single cell level. (d) Example of excitatory response evoked in an example TC (top). Gray bar indicates time of odor presentation. Scale bars = 20 mV, 0.4 s. (middle) Spike raster where timings of evoked APs are shown relative to a selected evoked spike (spike_ref) for 40 randomly chosen spike_ref. Spike autocorrelogram for this example cell is shown at the bottom (see methods). (e) as in (d) but with additional GCL silencing (yellow bar, top). Scale bars = 20 mV, 0.4 s. (f) Power spectra were obtained from the spike autocorrelogram for all TCs that showed excitatory responses (n = 9 cells from 8 animals) and averaged. Mean ± s.e.m are shown for odor only (black/gray) and with LED (green/pale green) conditions. Odors used = isoamylacetate, salicylaldehyde, eugenol, methylsalicylate and cinnamaldehyde at 2-5 % saturated vapor.

Figure 7. GCs contribute to fast, but not slow activities in awake animals

Whole cell and LFP recordings were made in the OB of awake, head-fixed Vgat-cre mice with AAV-FLEX-ArchT injection in the GCL (“GC silencing”). (a) Example whole cell recording from an infected GC that responded with AP discharge to stimulation with 5% of isoamylacetate. Odor-evoked responses under control conditions (top) and during light application (bottom). Scale bars, 20 mV, 1 s. (b) Spectral density estimates for an example LFP recording without external stimulus. Black = control, green = with LED. (c) Summary of average gamma power in the LFP (40-100 Hz). Mean ± s.e.m. shown. n = 11 recordings from 8 animals. (d,e) Effect of GC layer silencing on slow M/TC activities. (d) Effect of GC silencing on the baseline M/TC firing rates. Vm traces with repeated light presentations for an example cell (left). Scale bars = 10 mV, 0.5 s. Summary histogram of average firing rate change with light is shown on the right (FR_LED – FR_Control; n = 12 M/TCs from 5 animals). (right) Histogram shown with dotted line is for the baseline variability (FR_Control2 – FR_Control1; see Methods). (e) Effect of GC silencing on odor-evoked inhibition. (left) Example recordings from 2 cells where an odor evoked hyperpolarization during control (black traces), and the effect of GCL silencing (green traces). Scale bars = 5 mV, 0.5 s. Evoked Vm are summarized on the right; mean ± s.e.m. shown. (n = 8 cell-odor pairs from 3 animals). Odors used = isoamylacetate, salicylaldehyde, eugenol, methylsalicylate, acetophenone and ethylbutyrate at 1-3 % saturated vapor.