Excitatory actions of noradrenaline and metabotropic glutamate receptor activation in granule cells of the accessory olfactory bulb

Abstract

Modulation of dendrodendritic synapses by the noradrenergic system in the accessory olfactory bulb (AOB) plays a key role in the formation of memory in olfactory-mediated behaviors. We have recently shown that noradrenaline (NA) inhibits mitral cells by increasing gamma-aminobutyric acid inhibitory input onto mitral cells in the AOB, suggesting an excitatory action of NA on granule cells (GCs). Here, we show that NA (10 microM) elicits a long-lasting depolarization of GCs. This effect is mediated by activation of alpha(1)-adrenergic receptors as the depolarization is mimicked by phenylephrine (PE, 30 microM) and completely blocked by the alpha(1)-adrenergic receptor antagonist prazosin (300 nM). In addition to this depolarization, application of NA induced the appearance of a slow afterdepolarization (sADP) following a stimulus-elicited train of action potentials. Similarly, the group I metabotropic glutamate receptor (mGluR1) agonist DHPG (10-30 microM) also produced a depolarization of GCs and the appearance of a stimulus-induced sADP. The ionic and voltage dependence and sensitivity to blockers of the sADP suggest that it is mediated by the nonselective cationic conductance I(CAN). Thus the excitatory action resulting from the activation of these receptors could be mediated by a common transduction target. Surprisingly, the excitatory effect of PE on GCs was completely blocked by the mGluR1 antagonist LY367385 (100 microM). Conversely, the effect of DHPG was not antagonized by the alpha(1)-adrenergic receptor antagonist prazosin (300 nM). These results suggest that most of the noradrenergic effect on GCs in the AOB is mediated by potentiation of a basal activity of mGluR1s.

FIG. 1.

Noradrenaline (NA) directly excites granule cells (GCs). A, top trace: bath application of NA (10 μM, 2 min, top bar) produced a membrane potential depolarization and action potential firing in this GC. The responses evoked by NA had a slow onset (>40 s) and lasted several minutes (>10 min), after which the membrane potential returned to resting levels. Bottom trace: in the same cell, application of blockers of fast synaptic transmission [synaptic blockers; d-2-amino-5-phosphonopentanoic acid (APV), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), bicuculline (Bic)] and tetrodotoxin (TTX) to block voltage-gated Na channels, reduced the spontaneous synaptic activity but NA still induced a robust depolarization of GCs (resting membrane potential is −60 mV). B: in addition to membrane potential depolarization, NA (10 μM, 160 s, top bar) induced the appearance of a slow afterdepolarization (sADP) following a stimulus-induced train of action potentials. In this and the next figures GCs are stimulated every 30 s and the stimulus-elicited action potentials appear as upward lines due to compression in the time axis. Bottom traces: under control conditions (arrow 1 in top trace) the depolarizing current stimulus (30 pA, 500 ms) elicited 6 action potentials in this cell (inset). At the end of the stimulus the membrane potential was slightly hyperpolarized and then returned to resting levels. In the presence of NA (arrow 2, top trace) the same stimulus induced 8 action potentials (inset) and was followed by a small hyperpolarization; however, during the next seconds an sADP developed that greatly enhanced the excitation of the cell (see text for values and number of GCs tested). The dotted lines, in this and the following figures, indicate the resting membrane potential before the stimulus, −72 mV (1) and −65 mV (2).

FIG. 2.

The depolarization and sADP elicited by NA is due to activation of α₁-adrenergic receptors. A: the selective α₁-adrenergic receptor agonist phenylephrine (PE, 30 μM, 2 min, top bar) depolarized this GC and induced the appearance of an sADP following a stimulus-induced train of action potentials. During the application of PE the membrane potential was manually maintained at the resting value by passing negative current, thus counteracting the PE-induced depolarization. Nevertheless, a depolarizing stimulus (20 pA, 500 ms) still induced the sADP, which resulted in firing of the cell (bottom traces in expanded timescale; 1 and 2, control and in PE, respectively; membrane potential is −54 mV). B: the depolarization and ADP induced by NA are completely abolished by the selective α₁-adrenergic receptor antagonist prazosin. NA (10 μM, 150 s, top bar) depolarized this GC and induced the appearance of the sADP after a stimulus-induced train of action potentials (30 pA, 250 ms; insets 1 and 2: dotted line indicates the membrane potential and −63 and −55 mV, respectively). Bottom traces: in the same cell and in the presence of prazosin (300 nM) both the ADP (insets 1 and 2) and the depolarization induced by NA (10 μM, 150 s, top bar) are completely abolished.

FIG. 3.

Pharmacology and Ca²⁺ dependence of the NA-induced afterdepolarization. A: the depolarization and ADP induced by NA (10 μM, top bar; stimulus 47 pA, 500 ms) are completely abolished in the presence of the voltage-gated Ca²⁺ channel blocker cadmium (Cd²⁺, 200 μM, top left trace). In another cell the receptor-operated Ca²⁺ channel blocker SKF96365 (SKF, 30 μM, bottom left trace) also completely blocked the depolarization and ADP induced by NA (10 μM, top bar; stimulus 45 pA, 500 ms). B, top right traces: control and NA-induced sADP. Middle traces: in another GC recorded with the Ca²⁺ chelator BAPTA, the sADP induced by NA is completely abolished. Bottom traces: the response to NA is also completely abolished in the presence of the nonselective cation channel blocker flufenamic acid (FFA, 30 μM). C: graph bar summarizing the effects of different blockers on the depolarization and sADP elicited by NA (10 μM); both the depolarization and sADP are reduced by these blockers; the asterisks indicate a significance of ≥P < 0.01 (see text).

FIG. 4.

The sADP is voltage sensitive and Na dependent. A: the sADP induced by PE (30 μM, top left traces; stimulus 50 pA, 500 ms; 6 mV in this cell) is abolished when the extracellular Na concentration is reduced to 10 mM (bottom left traces; stimulus 50 pA, 500 ms). The calibration bar for the inset is 100 ms and 10 mV and the dotted line indicates the membrane potential before the depolarizing stimulus, control, and during PE (Control: −62 and −51 mV; low Na, −60 and −54 mV, respectively). The arrow indicates the AHP following the stimulus, which is not reduced in the low-Na solution. Left: graph bar summarizing the effects of reducing the extracellular Na concentration on the depolarization and sADP elicited by PE (30 μM); both the depolarization and sADP are reduced by reducing the Na; the asterisk indicates a significance of ≥P < 0.02 (see text). B, top: average current from subtracted current–voltage relationships obtained from a ramp under control conditions and in the presence of PE (30 μM; see methods). The PE-induced current was significantly larger at −40 than that at −100 mV (P < 0.01).

FIG. 5.

Metabotropic glutamate receptor (mGluR) activation depolarizes and elicits the appearance of the sADP in GCs. A, top traces: the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG, 10 μM, top bar) depolarized and induced the appearance of the slow ADP (inset: 1, control; 2 in DHPG). In the same cell the depolarization and stimulus-induced sADP elicited by PE (30 μM, top bar) has a time course similar to that elicited by DHPG (inset: 1, control; 2 in PE). The dotted lines indicate the membrane potential before the stimulus (top traces: −66 and −60 mV, 1 and 2, respectively; bottom traces: −62 and −57 mV, 1 and 2, respectively). B: graph bars showing the size (left), the time to peak (middle), and the duration (right) of the sADP elicited by the different agonists. These parameters were not significantly different for the sADP elicited by NA (10 μM), PE (30 μM), and DHPG (30 μM).

FIG. 6.

A group I mGluR antagonist abolished the excitatory response of PE in GCs. A: under control conditions PE (30 μM, top bar) depolarized and induced the sADP. In the presence of LY367385 (LY, 100 μM, upper top bar) the effect of PE was completely abolished. Bottom traces: expanded stimulus-induced responses, indicated by arrow and numbers in the top traces. In this cell the PE-induced sADP reached threshold and the cell fired (2); in the presence of LY, PE failed to induce the appearance of the sADP (4). The responses to the depolarizing stimulus in control and LY were not different (compare 1 and 3; in both A and B the stimulus is 50 pA, 500 ms). B: summary of the effect of blockers, LY effectively reduced the depolarizing response of PE (*P < 0.001). Conversely the depolarization induced by DHPG was not antagonized by the selective α₁-adrenergic receptor antagonist prazosin (300 nM).

FIG. 7.

The increase in the frequency of γ-aminobutyric acid (GABA) inhibitory postsynaptic currents (IPSCs) induced by PE in mitral cells is reduced by a mGluR1 antagonist. A: PE (30 μM, top bar) greatly increased the frequency of GABA IPSCs in mitral and tufted cells (MTCs) recorded in the presence of tetrodotoxin (TTX, 1 μM). Application of the mGluR1 antagonist LY (100 μM, long top bar) quickly reversed the PE-induced increase in frequency of IPSCs and a 2nd application of PE in the presence of LY completely failed to produce and increase in IPSCs. B: selected traces for mIPSCs for the same cell shown in A. The frequency but not the amplitude was increased in the presence of PE (see text). B: average cumulative interinterval distributions for the IPSCs (n = 4); control, gray line, circles; in the presence of PE, dark line, filled circles; in LY, gray line, empty squares; PE in the presence of LY, dark line, filled squares. Left: bar graph showing the effects of PE and LY on the mean frequency of IPSCs. PE significantly increased the frequency of IPSCs by about 10-fold (*P < 0.004). LY produced a small reduction in the mean frequency of IPSCs albeit not significantly (N.S., control vs. LY, P > 0.07). In the presence of LY PE failed to increase the frequency of IPSCs (see text for details). The holding potential in these experiments was 0 mV.