α(1A)-Adrenergic regulation of inhibition in the olfactory bulb

Abstract

By regulating inhibition at dendrodendritic synapses between mitral and granule cells (GCs), noradrenergic neurons extending from the brainstem provide an input essential for odour processing in the olfactory bulb (OB). In the accessory OB (AOB), we have recently shown that noradrenaline (NA) increases GABA inhibitory input on to mitral cells (MCs) by exciting GCs. Here, we show that GCs in the main OB (MOB) exhibit a similar response to NA, indicating a common mechanism for noradrenergic regulation of GCMC inhibition throughout the OB. In GCs of the MOB, NA (10 μM) produced a robust excitatory effect that included a slow afterdepolarization that followed a train of action potentials evoked by a current stimulus. The depolarization and slow afterdepolarization in GCs were blocked by the α1A-adrenergic receptor (AR) selective antagonist WB 4101 (30 nm) and mimicked by the α(1A)-AR selective agonist A 61603 (1 μM). In recordings from MCs, A 61603 (30 nm-1 μM) produced a sizeable increase in the frequency of spontaneous and miniature IPSCs, an effect completely abolished by the GABAA receptor antagonist gabazine (5 μM). Likewise, activation of β-ARs increased the frequency of spontaneous IPSCs; however, this effect was smaller and confined to the first postnatal weeks. NA enhanced inhibition in MCs across a broad concentration range (0.1-30 μM) and its effects were completely abolished by a mixture of α1- and β-AR antagonists (1 μM prazosin and 10 μM propranolol). Furthermore, the general α2-AR agonist clonidine (10 μM) failed to affect sIPSC frequency. Thus, the NA-mediated increase in GCMC inhibition in the OB results mostly from activation of the α1A-AR subtype.

Figure 1. NA increases GABA release in the MOB by exciting GCs

A, diagram of a sagittal slice of the OB showing the relative positions of the MOB and AOB and neurons we recorded from. LOT (dark line) forms a boundary that defines the AOB. Sensory neurons relay peripheral information via the ON and VN to GL in the MOB and AOB, where they synapse with MCs (dark grey). GCs (light grey) form numerous dendrodendritic synapses with MCs, influencing their output through GABAergic inhibition. Noradrenergic fibres innervate both the MOB and AOB where they are thought to regulate dendrodendritic synaptic activity. In the AOB, the LOT separates GCs from MCs. B, recording from a GC in the MOB where bath application of NA (10 μm) depolarized the cell to threshold and resulted in several minutes of spontaneous firing (−60 mV resting membrane potential). C, in another MOB GC, NA (10 μm, 100 s application) produced a slow afterdepolarization following a stimulus-induced train of action potentials (65 pA, 500 ms). Shown are the responses to current pulses before and after application of NA with expansions of the stimulus-elicited spike trains displayed in the insets. Similar to NA-induced excitatory responses in GCs of the AOB (see text and (Smith et al. 2009), the peak of this slow afterdepolarization (ΔV_m: 9 mV) occurred ∼3 s after the end of the stimulus pulse and had a slow decay. The dotted lines indicate the pre-stimulus membrane potential (control: −78 mV, NA: −68 mV; calibration bar: 3 s, 10 mV). D, top trace: recording from an MC in the MOB where NA (10 μm) dramatically increased the frequency of sIPSCs with a time course similar to that of the depolarization of GCs (control: 0.9 Hz, NA: 19.5 Hz). Bottom traces: representative sIPSCs from the above recording pre- and postapplication of NA (left and centre traces, respectively). This cell's sIPSCs were abolished by the GABA_A receptor antagonist GBZ (right traces; 5 μm). The holding potential was 0 mV in this and all following voltage clamp experiments. AOB, accessory olfactory bulb; GBZ, gabazine; GC, granule cells; GL, glomeruli, LOT, lateral olfactory tract; MC, mitral/tufted cells; MOB, main olfactory bulb; NA, noradrenaline; ON, olfactory nerve; sIPSC, spontaneous IPSCs; VN, vomeronasal nerve.

Figure 2. NA increases sIPSC frequency in MCs by activating α₁- and β-ARs in an age-dependent manner

A, left panel: recording of sIPSCs in an MC in the MOB of a P14 slice. Top traces: ISO (10 μm), a β-AR agonist, produced a ∼1-fold increase in sIPSC frequency (control: 0.5 Hz, ISO: 1.2 Hz). Bottom traces: the effect of PE (10 μm), an α₁-AR agonist, was much greater in the same cell, increasing sIPSC frequency by ∼15-fold (control: 0.6 Hz, PE: 10.2 Hz). Representative traces in an expanded time scale, before and after drug application, are shown below. Right panel: summary of the magnitude of the α₁- and β-AR-mediated effects on sIPSC frequency in MCs of the MOB. Though both ISO and PE increased the frequency of sIPSCs, the effect of PE was larger (*P < 0.00002; **P < 0.000001). The reported averages are from cells in P14–28 slices where both PE and ISO were applied (n= 21). B, left panel: recordings of sIPSCs from MCs in the AOB of slices from P4 (neonate, top traces) and P70 (adult, bottom traces) animals. While β-AR activation produced large increases in sIPSC frequency in young mice, ISO had no effect in older animals. Representative traces in an expanded time scale, before and after ISO application, are shown below. Right panel: summary of the effects of PE (black bars) and ISO (grey bars) on sIPSC frequency in the AOB of young and old mice. In both young (≤P7; n= 14) and old (≥P60; n= 10) mice, PE produced a significant increase in sIPSC frequency (**P < 0.0002). In contrast, in the same cells ISO significantly increased the frequency only in the younger group (*P < 0.05). AR, adrenergic receptor; AOB, accessory olfactory bulb; ISO, isoproterenol; MC, mitral/tufted cells; MOB, main olfactory bulb; PE, phenylephrine; sIPSC, spontaneous IPSCs.

Figure 3. NA excites GCs in the MOB and AOB by activating α_1A-ARs

A, top trace: recording from a GC in the MOB where bath application of NA (10 μm) depolarized the cell and produced an sADP that resulted in a series of spontaneous action potentials. In this cell, spike trains, which appear compressed as vertical lines in the time axis, were elicited every 30 s by a depolarizing current stimulus (30 pA, 500 ms). The right traces correspond to those indicated by the arrowheads and are shown in an expanded time scale. In these traces, the dotted lines indicate the pre-stimulus membrane potential. 1: under control conditions, the current stimulus produced seven spikes that were followed by a small afterhyperpolarization (ΔV_m: 1 mV). 2: post-application of NA, the same stimulus elicited 10 spikes and an sADP (7 mV) that greatly enhanced the cell's excitability. Bottom trace: in the same cell, application of a low concentration of the α_1A-AR selective antagonist WB 4101 (30 nm) completely abolished the depolarization and sADP produced by NA. 3 and 4: expanded responses to the current pulse at the points indicated with arrowheads (control and NA in the presence of WB 4101, the resting membrane potential is −62 mV). B, summary of the α_1A-AR-mediated changes in excitability of GCs in the MOB and AOB. The α_1A-AR selective antagonist WB 4101 (30 nm, white bars) greatly reduced the NA-induced depolarization (ΔV_m, black bars) in the MOB and AOB (n= 12 respectively). Likewise, application of WB 4101 significantly decreased the sADP in both regions (*P < 0.0006; MOB: n= 9; AOB: n= 6). Similarly, the NA-induced depolarization and sADP were mimicked by the α_1A-AR selective agonist A 61603 (1 μm, grey bars) in the MOB and AOB (n= 9, respectively). AOB, accessory olfactory bulb; AR, adrenergic receptor; GC, granule cells; MOB, main olfactory bulb; NA, noradrenaline; sADP, slow afterdepolarization.

Figure 4. α_1A-AR activation increases GABAergic inhibition on to MCs in the AOB and MOB

A, top trace: recording from an MC in the AOB, where sIPSC frequency was greatly increased by a low concentration of the α_1A-AR selective agonist A 61603 (30 nm; control: 2.1 Hz, A 61603: 8.0 Hz). Bottom traces: representative sIPSCs pre-application (left) and post-application (right) of A 61603 from the above trace. B, average cumulative inter-event interval distributions of sIPSCs in MCs of the AOB (left) and MOB (right). Application of 1 μm A 61603 (filled squares and circles) produced a leftward shift from the control event distribution (open squares and circles) consistent with an increase in sIPSC frequency. Insets: the average sIPSC amplitude in A 61603 (grey bars) was not different from control conditions (white bars) in the AOB (n= 7) or MOB (n= 8). AOB, accessory olfactory bulb; MCs, mitral cells; MOB, main olfactory bulb; sIPSCs, spontaneous IPSCs.

Figure 5. NA produces a concentration-invariant increase in sIPSC frequency in MCs

A, top traces: recording from an MC in the MOB where a low concentration of NA increased sIPSC frequency by ∼1-fold (control: 2.5 Hz, NA: 4.5 Hz). Lower traces: recording from an MC in the AOB where a low concentration of NA also increased sIPSC frequency (control: 2.5 Hz, NA: 4.5 Hz). Representative expanded traces pre- and post-application of NA are shown below. B, a low concentration of NA increased both sIPSCs (n= 10) and mIPSCs (n= 6) in the AOB. mIPSCs were recorded in the presence of dl-2-Amino-5-phosphonopentanoic acid, 6-Cyano-7-nitroquinoxaline-2,3-dione disodium and tetrodotoxin (**P < 0.05). C, dose–response curve for the effects of NA (black circles) and A 61603 (white squares) on sIPSC frequency in the AOB. Data were obtained from different cells and normalized to the fold increase in sIPSCs at the highest concentration of agonist. The fitted line was obtained using the Hill equation; A 61603 was more potent than NA (NA, EC₅₀: 6.9 μm; A 61603, EC₅₀: 0.1 μm). At 10 nm, A 61603 produced a small, though not significant, fold increase (control: 2.5 ± 1.0 Hz, A 61603: 4.3 ± 2.6 Hz, P > 0.3, n= 6). Similarly, 0.1 μm NA produced a small, but not significant, fold increase in frequency (control: 1.5 ± 0.5 Hz, NA: 1.9 ± 0.6 Hz, P > 0.1, n= 7). At concentrations greater than these, the frequency increases were statistically significant for both NA and A 61603 (see Results). mIPSCs, miniature IPSCs; MOB, main olfactory bulb; NA, noradrenaline; sIPSCs, spontaneous IPSCs.