Muscarinic receptor activation modulates granule cell excitability and potentiates inhibition onto mitral cells in the rat olfactory bulb

Abstract

The olfactory bulb is a second-order brain region that connects sensory neurons with cortical areas. However, the olfactory bulb does not appear to play a simple relay role and is subject instead to extensive local and extrinsic synaptic influences. Prime among the external, or centrifugal, inputs is the dense cholinergic innervation from the basal forebrain, which terminates in both the granule cell and plexiform layers. Cholinergic inputs to the bulb have been implicated in olfactory working memory tasks in rodents and may be related to olfactory deficits reported in people with neurodegenerative disorders that involve basal forebrain neurons. In this study, we use whole-cell recordings from acute rat slices to demonstrate that one function of this input is to potentiate the excitability of GABAergic granule cells and thereby modulate inhibitory drive onto mitral cells. This increase in granule cell excitability is mediated by a concomitant decrease in the normal afterhyperpolarization response and augmentation of an afterdepolarization, both triggered by pirenzepine-sensitive M1 receptors. The afterdepolarization was dependent on elevations in intracellular calcium and appeared to be mediated by a calcium-activated nonselective cation current (I(CAN)). Near firing threshold, depolarizing inputs could evoke quasipersistent firing characterized by irregular discharges that lasted, on average, for 2 min. In addition to regulating the excitability of the primary interneuronal subtype in the bulb, M1 receptors regulate the degree of adaptation that occurs during repetitive sniffing-like inputs and may therefore play a critical role in regulating short-term plasticity in the olfactory system.

Figure 1.

Cholinergic modulation of afterpotentials in granule cells. A, Schematic diagram of olfactory bulb. Labels indicate glomerular layer (Glom), external plexiform layer (EPL), mitral cell layer (MCL), and granule cell layer (GCL). We recorded from both granule cells (GC) and Blanes cells (BC) in different experiments in this study. B₁, CCh (2 μm) suppresses the afterhyperpolarization normally evoked by depolarizing steps in granule cells and reveals a BAPTA-sensitive afterdepolarization (arrow). Current step amplitude, 100 pA. The right panel is from a different experiment in which 10 mm BAPTA was added to the internal solution. The membrane potential is indicated below the trace. B₂, Summary of the effects of 2 μm CCh and BAPTA loading in 10 and 3 experiments, respectively. *p < 0.01. Amplitudes were measured 1 s after step. C₁, Carbachol had no effect on the afterdepolarization normally present in Blanes cells (100 pA step). C₂, Overlap of control and CCh traces from C₁. D, ADPs in granule cells are voltage dependent. Responses are averaged from three experiments in 2 μm CCh. Current step amplitudes were adjusted to generate 13–15 APs at each of the four membrane potentials (Mem. Pot.) tested. *p < 0.05. E₁, Blockade of ionotropic glutamate and GABA_A receptors with 5 μm NBQX, 25 μm d-APV, and 10 μm gabazine had no effect on the ADP response in granule cells treated with 2 μm CCh. Step amplitude, 50 pA. E₂, Summary of four experiments with the receptor antagonists shown in E₁. **p < 0.05. Action potentials are truncated in B, C, and E.

Figure 2.

Mechanism of afterdepolarization in granule cells. A₁, Cadmium (Cd; 200 μm) abolishes the afterdepolarization evoked in granule cells by 25 pA depolarizing steps in 2 μm CCh. A₂, Summary of the effect of Cd on ADP amplitude. *p < 0.01. B₁, Flufenamic acid (100 μm) blocks the ADP in CCh. B₂, Enlargement of CCh and CCh + FFA responses. Action potentials truncated in A₁ and B₂. C, ADPs evoked by Ca spikes in 2 μm CCh, 1 μm TTX, 25 mm TEA, 100 μm 4-AP and 4 mm CsCl. Reducing the Na driving force by applying an ACSF solution containing 99 mm NMG reversibly blocked the ADP (“+NMG”). Responses to 90 pA steps are truncated to illustrate afterpotentials. Example responses in CCh and CCh + NMG conditions shown in the insets. D, Thapsigarin (TG; 10 μm) did not reduce the ADP response in granule cells exposed to 2 μm CCh (50 pA step). E₁, Photolyzing NP-EGTA (2 mm; added to the internal solution) evokes a graded inward current in voltage-clamped granule cells. Horizontal lines above each trace indicate UV exposure time (100, 250, 500, and 1000 ms). E₂, UV exposure by itself does not evoke an inward current in control granule cells that were not loaded with NP-EGTA. F, Uncaging response reversed polarity at approximately −20 mV (data from 5 experiments). The inset shows example responses recorded at 0, −40, and −80 mV.

Figure 3.

Pharmacology of afterpotential modulation in granule cells. A, Left, Plot of the afterpotential response integral in CCh (2 μm) and after bath application of the M₁ mAChR antagonist pirenzepine (10 μm) and CCh. Pirenzepine abolishes the ADP response evoked by 50 pA depolarizing steps in CCh and restores the AHP normally present in granule cells under control conditions. Example traces shown above plot. Right, Bath application of the M₂ mAChR antagonist AF-DX 116 (1 μm; 50 pA step) does not block the ADP in granule cells. B, Bath application of the M₁ mAChR agonist MCN-A-343 (100 μm; 50 pA step) mimicked the effect of CCh and converted the normal AHP response into an ADP. Control and MCN-A-343 traces are shown overlapped on right. Action potentials truncated in the step responses in A and B are shown. All example responses in A are from approximately −55 mV; responses in B are from −50 mV. C, Summary of the effects of atropine and M₁ and M₂ receptor agents on the afterpotential integral in granule cells. *p < 0.01; **p < 0.0001.

Figure 4.

Differential effects of low and high concentrations of CCh on granule cell step responses. A, High concentrations of CCh (50 μm) increased the ADP amplitude (arrows) beyond the response recorded in 2 μm CCh and decreased the duration of firing during the step response (80 pA current step). Action potentials are truncated. B, Step responses from the traces in A shown at faster sweep speeds. C₁, Summary of the effect of low and high concentrations of CCh on granule cell input resistance. **p < 0.01. C₂, Summary of the effects of low and high concentrations of CCh on the amplitude of the first three action potentials in the step response. **p < 0.01. D₁, Fifty micromolars CCh increased the number of action potentials evoked by a relatively weak current step (60 pA). D₂, Summary of the effects of 2 and 50 μm CCh on granule cell responses to weak depolarizing steps (250 ms duration) that triggered 1–3 spikes in control. *p < 0.05; ***p < 0.001. E₁, Cell-attached recording from a spontaneously active granule cell in elevated KCl (5 mm instead of 3 mm) ACSF. Bath application of 2 μm CCh initially decreased AP amplitudes and then, at 3.25 min, abolished spiking. Spike frequency varied during CCh washing and occasionally became higher than in control conditions. Mean firing frequency is indicated above horizontal bars. Increasing bath CCh concentration to 50 μm did not recover spiking activity (data not shown). E₂, Response to 15 pA depolarizing current step after washout of CCh in whole-cell recording from the same neuron in E₁. E₃, Reconstruction of the granule cell recorded in cell-attached (E₁) and whole-cell (E₂) conditions from multiple maximum intensity projections of two-photon image z-stacks. Olfactory bulb layers are indicated above the montage.

Figure 5.

Muscarinic receptor activation prolongs granule cell responses to phasic excitatory input. A, Granule cell responses to a train of four simulated EPSPs (sEPSP; α function; tau = 100 ms) are increased in CCh (2 μm). The inset shows the effect of CCh on the step response recorded in the same granule cell. B, Bath application of 5 μm atropine reversed the facilitating effect of CCh. C₁, CCh significantly increased the total number of action potentials evoked by the four sEPSP train. This effect was abolished by 5 μm atropine. *p < 0.05. C₂, CCh also decreased the percentage of action potentials evoked by the first sEPSP and increased the percentage triggered by the last sEPSP. *p < 0.05; **p < 0.01. The number of action potentials calculated over windows of 400 ms aligned to the onset of each sEPSP. D₁, Bath application of 2 μm CCh reduced the latency granule cells discharge in response to a 5 Hz sine wave stimulus and decreased latency jitter. Control responses in black, responses in CCh shown in gray. D₂, Summary of 2 μm CCh-induced changes in AP latency and jitter (SD of latency) after 5 Hz sine wave stimulation in three experiments. *p < 0.05.

Figure 6.

Muscarinic receptor activation potentates synaptically evoked inhibition onto mitral cells. A₁, Cell-attached granule cell responses to a train of four glomerular layer stimuli (upward arrows; 400 ms interval; 100 μA) are increased after bath application of CCh (2 μm). Three consecutive responses shown in control conditions and in CCh. A₂, Mitral cell responses to a similar train of glomerular shocks also are potentiated by CCh. (GABA_A receptor-mediated currents are inward in these experiments because of the CsCl-based internal solution.) The inset shows most of the response to a single glomerular shock in CCh is blocked by 10 μm gabazine (different mitral cell than A₂). B, Summary of the selective potentiation of the late response (Stim 4; timing indicated solid bar above traces in A₂) by CCh with no significant increase in the early response (Stim 1; open bar) for both cell-attached granule cell recordings (B₁) and mitral cell intracellular responses (B₂). *p < 0.05. C, Cumulative plot of inhibitory responses in four mitral cells to glomerular shocks (Glom stim) before (dashed line) and after 2 μm CCh (solid line). D, Bath application of 2 μm CCh had no effect on mitral cell intrinsic physiology assessed by responses to slow, phasic stimuli (4 α functions, tau = 100 ms; interval, 400 ms) or depolarizing step stimuli (data not shown). E, Carbachol (2 μm) also had no effect on the barrage of glutamatergic EPSCs evoked in voltage-clamped granule cells after glomerular stimulation. Three consecutive responses are shown in each condition.

Figure 7.

Persistent activity in granule cells after muscarinic receptor activation. A, Depolarizing granule cells slightly (from −48 to −46 mV) converts the step-evoked ADP response into a prolonged action potential discharge. Responses to 50 pA steps recorded in 2 μm CCh. B, Persistent firing evoked by 50 pA depolarizing steps in CCh is abolished by the M₁ receptor antagonist pirenzepine (10 μm). Granule cell firing frequency was not constant and increased threefold during the response shown in the middle panel. C, Moderate intensity glomerular layer stimulation (4 × 100 μA; 400 ms interval) triggered spiking in a cell-attached granule cell recording. Bath application of CCh (2 μm) converted this transient response into a prolonged discharge that lasted >40 s. Gaps between consecutive sweeps shown were 20 s. D, Comparison of the persistent firing modes of granule cells (GC; top traces; recorded in 2 μm CCh; 80 pA step) and Blanes cells (BC; bottom traces; recorded under control conditions; 120 pA step). Both cells held at approximately the same membrane potential and stimulated with 500 ms depolarizing steps. Gaps between consecutive sweeps shown were 10 s for the GC and 20 s for the BC. The same voltage and time calibration for granule and Blanes cell recordings was used. E, Histograms of the interspike intervals in the response after the depolarizing steps (PF ISI) in the granule and Blanes cells shown in D. F, Summary of persistent firing frequency (PF Freq) and the coefficient of variation of the instantaneous firing frequency (PF CV) for granule cells in CCh and Blanes cells in control conditions. *p < 0.05. G, Summary of the differences between the RMP and persistent firing threshold (PF) for Blanes and granule cells.