Optogenetic activation of basal forebrain cholinergic neurons modulates neuronal excitability and sensory responses in the main olfactory bulb

Abstract

The main olfactory bulb (MOB) in mammals receives massive centrifugal input from cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, the activity of which is thought to be correlated with animal behaving states, such as attention. Cholinergic signals in the bulb facilitate olfactory discrimination and learning, but it has remained controversial how the activity of HDB cholinergic neurons modulates neuronal excitability and olfactory responses in the MOB. In this study, we used an optogenetic approach to selectively activate HDB cholinergic neurons and recorded the effect of this activation on the spontaneous firing activity and odor-evoked responses of mouse MOB neurons. Cells were juxtacellularly labeled and their specific types were morphologically determined. We find that light stimulation of HDB cholinergic neurons inhibits the spontaneous firing activity of all major cell types, including mitral/tufted (M/T) cells, periglomerular (PG) cells, and GABAergic granule cells. Inhibitions are significantly produced by stimulation at 10 Hz and further enhanced at higher frequencies. In addition, cholinergic activation sharpens the olfactory tuning curves of a majority of M/T cells but broadly potentiates odor-evoked responses of PG cells and granule cells. These results demonstrate strong effects of the basal forebrain cholinergic system on modulating neuronal excitability in the MOB and support the hypothesis that cholinergic activity increases olfactory discrimination capability.

Figure 1.

Optogenetic activation of HDB cholinergic neurons. A, Immunostaining against VAChT (red) verifies the cholinergic nature of ChAT–ChR2–EYFP⁺ neurons (green) in the HDB of ChAT–ChR2–EYFP mice. Arrows point to VAChT-immunopositive and EYFP⁺ neurons. Because of the membrane localization of ChR2 protein, EYFP signals tend to be weak in somata. B, Retrograde tracing confirms that the MOB receives input from HDB neurons, some of which are cholinergic. The fluorescent tracer Texas Red dextran amines were placed in the MOB to retrogradely label neurons in the HDB (red). Many tracer-labeled cells were EYFP⁺ (arrows). However, some of the labeled neurons lacked ChR2–EYFP expression, consistent with previous findings that the MOB is innervated by both cholinergic and non-cholinergic neurons in the HDB. C, Recordings from brain slices demonstrate that ChR2–EYFP⁺ neurons in the HDB can be activated by blue light stimulation with high temporal precision. The top panel shows that a brief pulse of blue light (5 ms; blue bar) elicited the firing of an action potential when the cell was recorded in the current-clamp mode (top trace) and an inward photocurrent in the voltage-clamp mode (bottom trace). The bottom panel shows that continuous light stimulation (500 ms; 20 mW/mm²; blue bar) resulted in depolarization and action potential firing in current-clamp mode (top trace) and a sustained photocurrent in voltage-clamp mode (bottom trace). D, Brief light pulses evoked reliable firing of action potentials at up to 50 Hz. E, Extracellular recordings from cells in the HDB of anesthetized mice illustrate that trains of light pulses (blue bars; 20 mW output from tip; 15 ms pulses; 50 Hz) produce vigorous firing of action potentials that tightly couples to the light stimulation. The left trace shows the recording from a representative HDB neuron, and the right trace displays a zoom-in view of the effect of 10 s light stimulation on neuronal firing.

Figure 2.

Activating HDB cholinergic neurons inhibits the spontaneous activity of M/T cells. A, A simplified schematic drawing shows the method of light stimulation of HDB cholinergic neurons and recordings from MOB neurons. An optical fiber was lowered with its tip above the HDB of ChAT–ChR2–EYFP mice. A glass pipette filled with Neurobiotin is used to record and fill neurons in the MOB. B, The morphology of a representative mitral cell, which was juxtacellularly labeled with Neurobiotin and visualized with Cy3–streptavidin. C, Physiological traces show that stimulating HDB cholinergic neurons resulted in suppression of spontaneous firing of a mitral cell in a frequency-dependent manner. The dashed line above the traces indicate light stimulation at the frequencies of 5, 10, 20, and 50 Hz (pulse duration of 15 ms). D, Population data on the effect of stimulating HDB at different frequencies on the spontaneous firing rates of M/T cells (n = 25 cells). Data are normalized to the mean spontaneous firing frequency within 30 s before light stimulation. In this and subsequent figures, error bars indicate SEM. E–G, Light stimulation at 50 Hz profoundly suppress the firing activity of M/T cells. E, Raw physiological trace (top) and the plot of mean firing rate (5 s per point) show that the cell shown in B is drastically inhibited after the optical activation (horizontal bar, 30 s) of HDB cholinergic neurons. F, Scatter plot of the mean firing rates before (horizontal axis) and during 30 s, 50 Hz light stimulation (vertical axis) of HDB cholinergic neurons. Each dot represents an individual M/T cell. Dashed line indicates no change of firing rates in spontaneous activity after light stimulation. Arrow points to the cell shown in B. All 25 M/T cells exhibit a reduction of spontaneous firing in response to 50 Hz light stimulation. G, Bar graph shows that 50 Hz light stimulation produces a 84% reduction in spontaneous firing rates. ***p < 0.001, paired t test; n = 25 M/T cells. Ctrl, Control.

Figure 3.

The response tuning of M/T cells is sharpened by the activity of HDB cholinergic neurons. A, Scatter plot of response strengths during light stimulations at 10 Hz (left) and 50 Hz (right) against the same during control conditions. Dots represent individual odor–M/T cell pairs. Dashed line indicates no change in response strength following light stimulation. B, Bar plot of the mean response strength of M/T cells before and during light stimulation. n.s., Statistically not significant. ***p < 0.001, paired t test; n = 400 odor–cell pairs. Ctrl, Control. C, The distribution of the variance indexes of 25 M/T cells. Each dot represents an individual M/T cell to light stimulation at 50 Hz (blue) or 10 Hz (red). Cells were aligned by values of variance indexes at 50 Hz. The variance index of an individual cell was calculated by averaging the absolute difference of odor-evoked responses before and during light stimulation. D, Mean variance index for 10 and 50 Hz stimulations. **p < 0.01, paired t test; n = 25 cells. E, Olfactory tuning curves of four M/T cell before and during laser stimulation at 10 or 50 Hz. Numbers represent changes of firing rates (spike/s; ΔHz) induced by odorant application, with positive ones indicating excitation and negative ones inhibition. F, Sample raw traces (top panels) and PSTHs (bottom panels) show diverse effects of activating HDB cholinergic neurons on odorant-evoked responses of the cell. Italic letters a and b correspond to the data values indicated by arrows in E₁. Light stimulation of cholinergic neurons enhanced the excitatory response to the optimal odorant (a) but reduced the response to a non-optimal odorant (b). Black horizontal bars indicate 2 s odorant application and blue bars indicate light stimulation.

Figure 4.

HDB cholinergic activation could enhance inhibitory responses or produce odor-specific modulations for a subset of M/T cells. A, B, The olfactory tuning curves (A) and representative traces (B) of a mitral cell show increased inhibition after stimulation of HDB cholinergic neurons at 10 or 50 Hz. Ctrl, Control. C, D, The tuning curves and traces of a broadly responsive mitral cell. Light stimulation at 10 Hz did not produce strong effects on tuning, but stimulation at 50 Hz resulted in enhanced excitation of several odorants. Same conventions as Figure 3, E and F.

Figure 5.

The spontaneous firing activity of PG cells is suppressed by stimulating HDB cholinergic neurons. A, The morphology of a representative PG cell. B, C, Physiological traces (B) and normalized spontaneous firing rates (C) show that HDB stimulation inhibits the firing activity of PG cells in a frequency-dependent manner. The curve in C includes eight PG cells that were inhibited by light stimulation. **p < 0.01, ***p < 0.001, paired t tests between control and light stimulation at respective frequencies. D, Light stimulation of HDB cholinergic neurons at 50 Hz completely silenced spike firing of a PG cell. E, Scatter plot reveals that the spontaneous firing activity of a vast majority of PG cells (8 of 9) was reduced by activating HDB cholinergic neurons. F, Bar graph shows that 50 Hz light stimulation significantly reduces spontaneous activity of PG cells (*p < 0.05, paired t test; n = 9 cells). Ctrl, Control.

Figure 6.

Light stimulation of HDB cholinergic neurons potentiates the olfactory responses of PG cells. A, The odor-evoked responses of PG cells are increased by light stimulation of HDB cholinergic neurons at 10 Hz (1) or 50 Hz (2). B, Bar graph shows a significant increase in the mean response strength of PG cells during light stimulation, and higher frequency stimulation leads to a stronger potentiatory effect on odor responses. ***p < 0.001, paired t test; n = 160 odor–cell pairs. Ctrl, Control. C, D, The tuning curves (C) and physiological traces (D) illustrate that activating HDB cholinergic neurons produces a broad increase in the excitatory responses of a PG cell. E, F, The olfactory tuning curves (E) and representative traces (F) of a PG cell show non-uniformed potentiation by light stimulation. Ctrl, Control.

Figure 7.

Stimulating HDB cholinergic neurons inhibits the spontaneous firing activity of granule cells. A, The morphology of a granule cell. B, C, Physiological traces (B) and plot of normalized firing rates (C) show the frequency-dependent inhibition of granule cells by the stimulation of HDB cholinergic neurons. The curve in C excludes data from one granule cell that was not inhibited by cholinergic activation. ***p < 0.001, paired t tests between control and light stimulation at different frequencies; n = 15 cells. D, The physiological trace and plot of mean firing rate of a granule cell that exhibited a reduction in spontaneous activity after 50 Hz light stimulation. E, Scatter plot illustrates that the spontaneous firing activity of a vast majority of granule cells (15 of 16) was reduced by HDB stimulation. F, Population data show that 50 Hz light stimulation results in a significant reduction in the mean spontaneous activity of granule cells (**p < 0.01, paired t test; n = 16 cells). Ctrl, Control.

Figure 8.

Activating HDB cholinergic neurons enhances the olfactory responses of granule cells. A, Scatter plots show the potentiatory effects on odor-evoked responses of granule cells by stimulating HDB at 10 Hz (1) or 50 Hz (2). B, Bar plots of olfactory response strength before and during 10 or 50 Hz light stimulation. ***p < 0.001, paired t test; n = 288 odor–cell pairs. Ctrl, Control. C, D, The tuning curves (C) and raster plot (D) illustrate that activating HDB cholinergic neurons broadly increased olfactory responsiveness of a granule cell. The raster plot in D displays the firing activity of a total of 80 trials for 16 odorants. Trials were aligned along the vertical axis, and each action potential is plotted as a black dot along the horizontal axis. E, F, The olfactory tuning curves (E) and representative traces (F) of a granule cell show odorant-specific potentiation of response strength by HDB stimulation.