Adrenergic enhancement of inhibitory transmission in the accessory olfactory bulb

Abstract

Noradrenergic modulation of dendrodendritic synapses between the mitral and granule cells in the accessory olfactory bulb (AOB) is postulated to play a key role in the formation of memory in olfactory-mediated behaviors. Current models propose that noradrenaline (NA) increases excitation of mitral/tufted cells (M/TCs) by decreasing the release of GABA from granule cells. However, surprisingly little is known about the actions of NA at the cellular level in the AOB. Here, in recordings from AOB slices, we show that NA decreases the firing frequency of M/TCs in response to stimulation. This effect is attributable to an increase in the GABA inhibitory input to M/TCs. Application of NA (10 microM) produced an approximately 20-fold increase in the frequency of GABA-induced miniature IPSCs (mIPSCs) without changing their amplitude. A pharmacological analysis indicated that the increase in mIPSCs frequency results from activation of alpha1 adrenergic receptors. In addition to increasing the mIPSC frequency, NA also potentiated GABA inhibitory currents induced by direct stimulation of granule cells. Together, our results suggest that NA increases the release of GABA from granule cells by acting on presynaptic receptors. Thus, the role of the noradrenergic activity in the AOB may be opposite than suggested previously: we find that the overall effect of NA in the AOB is inhibition of M/TCs.

Figure 1.

Noradrenaline decreases the frequency of firing of M/TCs in cell-attached patches. A, Bath application of NA (10 μm, 2 min) reduced the frequency of firing in M/TCs that were spontaneously active (control, 1.2 Hz; after NA, 0.5 Hz in this cell). The effect of NA has a slow onset (>40 s). B, The average frequency of firing was significantly reduced after the application of NA (control, 2.4 ± 0.6 Hz; after NA, 0.8 ± 0.4 Hz; *p < 0.006; n = 5). C, The size of the spikes was increased after the application of NA, suggesting that the cell hyperpolarized (selected spikes from the cell shown in A). The pipette holding potential was 0 mV.

Figure 2.

Noradrenaline decreases the frequency of firing during a depolarizing current pulse. A, NA produced a small hyperpolarization of M/TCs, 2.5 mV in this cell; the dotted line represents the resting membrane potential (−61 mV). The vertical lines are compressed trains of spikes (see below). B, Cells were stimulated with a depolarizing current pulse (100 pA, 500 ms). NA decreased the number of spikes elicited by the stimulus. Notice the small afterhyperpolarization at the end of the depolarizing pulse attributable to dendrodendritic inhibition, which was increased by NA in this cell. Inset, NA also increased the synaptic noise (this is the same cell shown in A). C, The firing frequency was decreased in seven of eight cells tested. Average control, 24.4 ± 2.4 Hz (○); after NA, 18.6 ± 3.2 Hz (□); p < 0.01, one-tailed t test.

Figure 3.

Noradrenaline increases the frequency of GABA mIPSCs. A, NA (10 μm, 2 min) greatly increased the frequency of mIPSC in M/TCs recorded in the presence of TTX (1 μm), NBQX (10 μm), and APV (100 μm). Calibration: 20 pA, 20 s. B, Selected traces for mIPSCs for the same cell shown in A. NA increased both the small- and large-amplitude mIPSCs (see Results). The effect of NA was completely blocked by the GABA_A receptor antagonist bicuculline methiodide (BMI; 20 μm). C, Average cumulative amplitude (left) and inter-interval distributions (right) for the mIPSCs, control (gray) and in the presence of NA (dark thick line; n = 6). Left, The cumulative amplitude shows a small shift to the left attributable to an increase in the frequency of small-sized mIPSCs. However, the average amplitude was not changed (control, 20.8 ± 2.1 vs 17.1 ± 1.9 pA in NA; inset bar graph). Right, The cumulative interevent distribution shows a shift to the left in the presence of NA resulting from the increase in frequency of mIPSCs (0.4 ± 0.1 vs 10.8 ± 1.1 Hz; inset bar graph, *p < 0.0002). The holding potential was 0 mV.

Figure 4.

Noradrenaline increases the frequency of GABA mIPSCs by activating α1 adrenergic receptors. A, The α1 adrenergic agonist PE (30 μm) mimics the effect of NA. Neither the α2 adrenergic agonist clonidine (CLO; 10 μm) nor the β adrenergic agonist isoproterenol (ISO; 10 μm) modified the frequency of mIPSC. The α1 adrenergic antagonist prazosin (PRA; 100 nm) completely blocked the effect of NA. The PE, clonidine, and isoproterenol records shown are from the same cell. B, The control average mIPSC frequency (0.9 ± 0.2 Hz; n = 15) was greatly increased by NA (8.8 ± 2.3 Hz; n = 4; *p < 0.001) and by PE (8.5 ± 2.2 Hz; n = 4; *p < 0.001), although it was unchanged by clonidine or isoproterenol (0.5 ± 0.1 and 1.3 ± 0.8 Hz, respectively; both n = 3). The effect of NA was completely blocked by PRA (0.5 ± 0.2 Hz; n = 4). The holding potential was 0 mV.

Figure 5.

Noradrenaline increases the glutamate-evoked release of GABA. A, A glutamate pulse (1 mm, 100 ms, every 30 s) was used to stimulate granule cells. The glutamate-containing patch pipette was positioned in the granule cell layer, in the vicinity of the recorded M/TC. The glutamate pulse produced a barrage of GABA IPSCs, which were enhanced by NA (10 μm, 2 min). In this figure, the gap (20 s) between consecutive glutamate applications has been removed to better illustrate the effect of NA. B, Average currents for glutamate-induced GABA (see below) currents before and after NA (7 pulses each) for the cell shown in A. Both the baseline and the glutamate-induced IPSCs frequency were enhanced in the presence of NA. C, The glutamate-evoked IPSCs are completely blocked the GABA_A receptor antagonist bicuculline (BMI; 20 μm; n = 4). D, The percentage increase in current induced by glutamate was higher in the presence of NA (control, 35 ± 7%; after NA, 69 ± 13%; *p < 0.003, one-tail t test; n = 6). The holding potential was −60 mV.

Figure 6.

The noradrenaline-induced increase in mIPSC frequency is dependent on calcium ions. A, Effect of NA (10 μm, 2 min) on mIPSC frequency in control (top) and in the presence of the calcium channel blockers Cd²⁺ and Ni²⁺ (bottom; each at 100 μm). In control conditions, NA greatly increased the frequency of mIPSC (before, 0.4 Hz; after NA, 4.2 Hz). In the presence of Cd²⁺ and Ni²⁺, the effect of NA was reduced (before, 0.2 Hz; after NA, 0.8 Hz). B, The average mIPSC frequency after NA was significantly increased in control conditions (before, 0.8 ± 0.4 Hz; after NA, 7.7 ± 1.9; *p < 0.02; n = 5), although it was not different in the presence of Cd²⁺ and Ni²⁺ (before, 0.5 ± 0.1 Hz; after NA, 1.3 ± 0.7 Hz; p > 0.3). The holding potential was 0 mV.

Figure 7.

Activation of α1 adrenergic receptors depresses the input from vomeronasal nerve. A, Action potentials were evoked by stimulation of the VN layer (see Materials and Methods). Application of PE (30 μm, 2.5 min) produced a long-lasting decrease in the number of stimulus-evoked action potentials. Inset, In the same cell, the number of spikes produced by a depolarizing current is reduced (500 ms, 50 pA). Bottom row, Prazosin (300 nm) completely blocked the inhibitory effect of PE on the action potentials evoked by VN stimulation, as well as the activity in response to the intracellular depolarizing current (inset calibration: 1 s, 20 mV). B, Time course of the effect of PE on action potentials evoked by stimulation in the vomeronasal nerve layer. PE (30 μm, 2.5 min) produced a long-lasting, reversible depression in the number of action potentials evoked by VN stimulation. This effect was completely blocked by 300 nm prazosin (applied ∼20 min; dotted line).