Adenosine A1 receptor activates background potassium channels and modulates information processing in olfactory bulb mitral cells (2024)

• Adenosine is a widespread neuromodulator in the mammalian brain, but whether it affects information processing in sensory system(s) remains largely unknown.

• Here we show that adenosine A₁ receptors hyperpolarize mitral cells, one class of principal neurons that propagate odour information from the olfactory bulb to higher brain areas, by activation of background K⁺ channels.

• The adenosine‐modulated background K⁺ channels belong to the family of two‐pore domain K⁺ channels.

• Adenosine reduces spontaneous activity of mitral cells, whereas action potential firing evoked by synaptic input upon stimulation of sensory neurons is not affected, resulting in a higher ratio of evoked firing (signal) over spontaneous firing (noise) and hence an improved signal‐to‐noise ratio.

• The study shows for the first time that adenosine influences fine‐tuning of the input–output relationship in sensory systems.

Ethical approval

Animal rearing, dissection and all experiments were performed according to the European Union's and German animal welfare guidelines and were approved by the local authorities (GZ G21305/591‐00.33; Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany). They conform to the principles and regulations described by Grundy (2015). Mice of both sexes (age: postnatal days 8–21) were anaesthetized with isoflurane and decapitated.

Animals and preparation procedure

Naval Medical Research Institute (NMRI) outbred mice, OMP‐synapto‐pHluorin‐expressing mice (Bozza etal. 2004) and A₁ receptor knockout mice (Johansson etal. 2001) were bred in the institute's animal facility (12h–12h light–dark cycle, food and water ad libitum). Olfactory bulbs were dissected and horizontal brain slices were prepared as described before (Fischer etal. 2012). Olfactory bulbs were quickly transferred into a chilled slicing artificial cerebrospinal fluid (ACSF) that contained (in mm): NaCl, 83; NaHCO₃, 26.2; NaH₂PO₄, 1; KCl, 2.5; sucrose, 70; d‐glucose, 20; CaCl₂, 0.5; MgSO₄, 2.5. Sagittal slices 200μm thick of the bulbs were cut using a vibrating blade microtome (Leica VT1200S, Bensheim, Germany). Brain slices were stored in ACSF containing (in mm): NaCl, 120; NaHCO₃, 26; NaH₂PO₄, 1; KCl, 2.5; d‐glucose, 2.8; CaCl₂, 2; MgCl₂, 1. Storage lasted for 30min at 30°C and 15min at room temperature before starting experiments. ACSF was continuously gassed with carbogen (95% O₂–5% CO₂; buffered to pH7.4 with CO₂/bicarbonate).

Drugs

Adenosine, adenosine‐5′‐diphosphate, adenosine‐5′‐triphosphate, barium chloride, halothane and 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) were purchased from Sigma‐Aldrich (Munich, Germany); N⁶‐cyclopenthyladenosine (N⁶‐CPA), N‐[4‐[[1‐[2‐(6‐methyl‐2‐pyridinyl)ethyl]‐4‐piperidinyl]carbonyl]phenyl]‐methanesulfonamide dihydrochloride (E‐4031), tetrodotoxin citrate (TTX), 10,10‐bis(4‐pyridinylmethyl)‐9(10H)‐anthracenone dihydrochloride (XE 991), 6,12,19,20,25,26‐hexahydro‐5,27:13,18:21,24‐trietheno‐11,7‐metheno‐7H‐dibenzo [b,n]‐[1,5,12,16]tetraazacyclotricosin‐5,13‐diium dibromid (UCL 1684) and 4‐aminopyridine (4‐AP) were from Tocris (Bristol, UK); 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo‐[f]‐quinoxaline‐7‐sulfonamide (NBQX), d‐(−)‐2‐amino‐5‐phosphonopentanic acid (d‐APV) and gabazine hydrobromide were from Abcam (Cambridge, UK); tetramethyl ammonium (TEA) was from Applichem (Darmstadt, Germany); 1‐[(2‐chlorophenyl)diphenylmethyl]‐1H‐pyrazole (TRAM‐34) was from Alomone Labs (Jerusalem, Israel).

Electrophysiological recordings

Mitral cells of the main olfactory bulb were investigated using the patch‐clamp technique (MultiClamp 700B amplifier and pCLAMP 10 software, Molecular Devices, Biberach, Germany). Throughout the experiments, brain slices were superfused with ACSF. In ACSF with 5mm K⁺ concentration, equimolar amounts of NaCl were replaced by KCl. Drugs were applied with the perfusion system driven by a peristaltic pump (Reglo, Ismatec, Wertheim, Germany) at a flow rate of 2mlmin⁻¹. Application of drugs was achieved by manually changing the inflow of the pump from drug‐free to drug‐containing ACSF. Application bars reflect the time window during which drug‐containing ACSF was added to the bath, the time being corrected for the time needed for the drug to pass the tubing and enter the bath. Complete bath exchange required approximately 30s. If not stated otherwise, the whole‐cell configuration was employed using patch pipettes with a resistance of ∼3MΩ. Recordings were digitized (Digidata 1440A, Molecular Devices, Biberach, Germany) at 10–20kHz and filtered (Bessel filter, 2kHz). The standard pipette solution contained (in mm): NaCl, 10; potassium gluconate, 105; K₃‐citrate, 20; Hepes, 10; MgCl₂, 0.5; Mg‐ATP, 5; Na‐GTP, 0.5; mannitol, 21; phosphocreatine, 5. Pipette solution with reduced K⁺ concentration contained (in mm): NaCl, 10; K‐gluconate, 110; K₃‐citrate, 10; Hepes, 10; EGTA, 0.25; MgCl₂, 0.5; Mg‐ATP, 3; Na‐GTP, 0.5. High‐chloride pipette solution contained (in mm): NaCl, 10; potassium gluconate, 52.5; KCl, 55; K₃‐citrate, 10; Hepes, 10; EGTA, 0.25; MgCl₂, 0.5; Mg‐ATP, 3; Na‐GTP, 0.5. The pipette solutions contained 8μm Alexa Fluor 594 in most of the experiments. For extracellular recordings of action potentials, patch pipettes were filled with ACSF (resistance ∼3MΩ) and the cell‐attached configuration was established. For stimulation of axons of olfactory sensory neurons we used ACSF‐filled pipettes with a resistance of ∼2MΩ. The stimulation pipette was placed in the olfactory nerve layer at a distance of at least 50μm to the apical dendrite of the recorded mitral cell. Current pulses (100μA, 50μs) were applied to evoke synaptic excitation of mitral cells. In control experiments, synaptic currents were entirely blocked by application of 1μm TTX, confirming that stimulation‐induced currents were synaptically evoked, but not evoked by direct stimulation of the mitral cell.

Measurements of membrane conductance and reversal potential

The membrane conductance was measured in voltage clamp with a holding potential of −70mV. A hyperpolarizing voltage step of 10mV and 1s duration was applied every 10s and the membrane conductance was calculated from the resulting current shift that was measured at the end of the voltage step. To estimate the reversal potential of the adenosine‐induced membrane potential shift, the membrane potential was recorded in current clamp and hyperpolarized by current steps of 500ms duration and increasing amplitude (50pA interval) before and during application of adenosine. The difference between membrane potential values recorded before and during adenosine application at a given current step was calculated, and the values of all experiments averaged and plotted against the membrane potential value before adenosine application. All data were pooled and a linear regression was calculated. The point of intersection of the regression line and the abscissa reflects the reversal potential of the adenosine‐induced membrane potential shift. The current–voltage relationship of the adenosine‐evoked current was obtained by applying voltage ramps (duration: 800ms) from −20 to −130mV in the absence and in the presence of adenosine. The difference of the obtained current traces revealed the adenosine‐evoked current. The resulting current–voltage relationship was corrected for a liquid junction potential of −18mV.

Synapto‐pHluorin recordings

Mice expressing synapto‐pHluorin (syn‐pH) exclusively in olfactory sensory neurons were employed to study the effect of adenosine on vesicle fusion in olfactory sensory nerve terminals as described in Thyssen etal. (2010). Olfactory bulbs in toto (Stavermann etal. 2015) were placed under a confocal microscope (eC1, Nikon, Düsseldorf, Germany) and syn‐pH was excited at 488nm. Fluorescence was collected using a bandpass filter between 515 and 545nm. Olfactory sensory axons were stimulated using a stimulation isolator (DS3, Digitimer, Welwyn Garden City, UK). Regions of interest were defined in the glomeruli and changes in the syn‐pH fluorescence were calculated with the baseline fluorescence before stimulation taken as 100%.

A₁ receptor in situ hybridization

Data analysis and statistics

Patch‐clamp recordings were analysed using Mini Analysis (Synaptosoft, Fort Lee, NJ, USA), ClampFit (Molecular Devices, Biberach, Germany) and OriginPro (OriginLab Corp., Northampton, MA, USA). Membrane potential values recorded in current clamp using a low‐chloride pipette solution were corrected for a liquid junction potential of −18mV. All values are means ± standard error of the mean (SEM) with n representing the number of analysed cells. Statistical analysis was performed using one‐way ANOVA with Fisher's post hoc test or Student's t test. Means were defined as statistically different at error probabilities P<0.05 (^*), P<0.01 (^**) and P<0.001 (^***).

Adenosine A₁ receptor gene expression in olfactory bulb mitral cells

We first assessed the expression profile of the A₁ adenosine receptor gene (ADORA1) in parasagittal sections of the mouse brain. As described before (Mahan etal. 1991; Reppert etal. 1991), we detected high levels of A₁ receptor mRNA in hippocampus and cerebellum. In addition, we found A₁ receptor expression in the olfactory bulb (Fig.1A). One of the strongest expression signals of A₁ in the entire brain was found in the olfactory bulb mitral cell layer. At higher magnification, it became evident that the expression signal in the mitral cell layer correlated to large cell bodies of mitral cells (Fig.1B and C). In the external plexiform layer and the basal part of the glomerular layer, cell bodies presumably reflecting tufted and external tufted cells were labelled. The expression of A₁ receptor gene, as assessed by in situ hybridization, was very low in the outer part of the glomerular layer and the granule cell layer. A1 adenosine receptor expression was not observed using the sense probe and in A1 receptor knockout mice using the antisense probe, confirming the specificity of the in situ hybridization probes (Fig.1D–G).

Expression of the A_2A adenosine receptor gene (ADORA2A) was most prominent in the caudate putamen and the olfactory bulb (Dixon etal. 1996; Svenningsson etal. 1997; Kaelin‐Lang etal. 1999). The expression of A_2A in the olfactory bulb was more widespread compared to A₁ receptors and was detected in cells of all layers of the olfactory bulb, including mitral cells (Fig.1H–J). Hence, high levels of adenosine receptor expression were found in the olfactory bulb and in particular in mitral cells, emphasizing the possible role of adenosine as a neuromodulator in this brain area.

Activation of adenosine A₁ receptors hyperpolarizes mitral cells

Physiological data suggest that adenosine might play a role in modulation of neuronal performance in the olfactory bulb (reviewed in Lohr etal. 2014). Therefore, we tested the effect of adenosine on the membrane potential of mitral cells. Mitral cells of wild‐type mice had a mean resting membrane potential of −63.0±0.6mV (n=23), which hyperpolarized by 2.6±0.4mV (n=35) upon application of 100μm adenosine for 30s (Fig.2A, left panel). The membrane potential recovered slowly to resting values, presumably due to the slow bath perfusion kinetics. In addition, we cannot exclude the involvement of a sustained component in the adenosine‐stimulated signalling pathway leading to hyperpolarization. In A₁ receptor knockout mice, mitral cells did not hyperpolarize due to adenosine application, but instead depolarized by 0.4±0.2mV (n=9, P=0.005), suggesting that A₁ receptors mediate the adenosine‐evoked hyperpolarization (Fig.2A, right panel). The hyperpolarizing effect of adenosine in wild‐type mice was mimicked by application of the A₁ agonist N⁶‐CPA (Fig.2B). 1μm of N⁶‐CPA evoked a hyperpolarization of 2.8±0.4mV (n=13). In the presence of the potent and selective A₁ receptor antagonist DPCPX (1μm), no change in membrane potential was evoked by adenosine (n=6), confirming that adenosine hyperpolarized mitral cells by activation of A₁ receptors (Fig.2C). DPCPX alone had no effect on the resting membrane potential and the resting firing rate of mitral cells. The pharmacological profile of the adenosine‐mediated effect on mitral cells, summarized in Fig.2D, clearly demonstrates an activation of A₁ receptors by adenosine. Previous studies demonstrated release of ATP from olfactory sensory neurons and its degradation to adenosine in glomeruli (Doengi etal. 2008; Thyssen etal. 2010), leading to the hypothesis that the adenosine‐mediated hyperpolarization in mitral cells mainly originates from the apical tuft in the glomerulus. Therefore, we compared the hyperpolarization evoked by adenosine between mitral cells with intact apical dendrite and mitral cells in which the apical tuft had been cut during preparation of brain slices, as visualized by the fluorescent tracer Alexa Fluor 594 added to the pipette solution. We did not find a significant difference in the adenosine‐evoked hyperpolarization between these groups (Fig.2E). Adenosine evoked a hyperpolarization of 2.6±0.4mV (n=20) in intact mitral cells and 2.6±0.7mV (n=15) in mitral cells with cut apical tuft, indicating that the adenosine‐evoked hyperpolarization does not mainly derive from the apical tuft.

A₁ receptor activation increases the membrane conductance in mitral cells

The adenosine‐evoked hyperpolarization via A₁ receptors could be caused either by the closure of depolarizing non‐specific cation channels or by an opening of hyperpolarizing Cl⁻ or K⁺ channels. To assess whether channels are opened or closed by application of adenosine, we measured the membrane conductance in voltage‐clamped mitral cells (holding potential: −70mV) in the presence of the glutamate receptor antagonists NBQX (10μm) and d‐APV (50μm), the GABA_A receptor antagonist gabazine (10μm) and TTX (1μm) to synaptically isolate the recorded mitral cell from its neuronal network. Membrane conductance was assessed by applying voltage steps of −10mV amplitude. Application of adenosine led to an outward current (Fig.3A) and an increase in membrane conductance (Fig.3B). Interestingly, the increase in membrane conductance outlasted the outward current and did not reach baseline within 5min after washout of adenosine (Fig.3C). On average, the membrane conductance significantly increased by 21.4±4.4% from 3.24±0.8 to 3.55±0.8nS (n=6; P<0.001) upon application of adenosine, indicating that A₁ receptors in mitral cells are linked to the opening of ion channels that mediate hyperpolarization such as Cl⁻ or K⁺ channels.

We tested the involvement of Cl⁻ channels by using a modified pipette solution in order to shift the reversal potential for Cl⁻. The calculated Cl⁻ reversal potential using the standard (low Cl⁻) pipette solution was −61mV, which is close to the resting membrane potential. We used a high Cl⁻ pipette solution resulting in a calculated Cl⁻ reversal potential of −16mV. Hence, under the latter conditions, opening of Cl⁻ channels should have led to depolarization. However, application of adenosine elicited a hyperpolarization, regardless of which pipette solution was used (Fig.3D). The amplitudes of the hyperpolarizations measured with different pipette solutions were not significantly different, suggesting that Cl⁻ channels make no major contribution to the adenosine‐mediated hyperpolarization (Fig.3E).

A₁ receptors activate K⁺ channels

We determined the reversal potential of the adenosine‐mediated membrane potential shift in current‐clamp by stepping to different membrane potentials using the standard pipette solution and defined current steps (50pA interval) in the absence and in the presence of adenosine (Fig.4A). We used a combination of K⁺ concentrations in the extracellular (5mm) and pipette solution (140mm) that resulted in a calculated K⁺ reversal potential of −86mV. On average, at membrane potentials more positive than −90mV, adenosine tended to hyperpolarize the membrane, whereas at membrane potentials more negative than −90mV, adenosine depolarized the membrane (Fig.4B). The reversal potential of the adenosine‐mediated shift in membrane potential as extrapolated from all recordings (n=5 cells) was −90mV, close to the calculated K⁺ reversal potential.

To confirm that adenosine activates a K⁺ conductance, we measured the ionic current evoked by adenosine in voltage‐clamp experiments at different holding potentials. In these experiments the K⁺ reversal potential was set to −108mV to increase the driving force for K⁺ and, hence, the size of the responses evoked by adenosine. To assess the current–voltage relationship, the membrane potential was first clamped to −20mV for several minutes to inactivate voltage‐dependent currents, followed by a voltage ramp from −20 to −130mV within 800ms. Voltage ramps were performed before and during application of 100μm adenosine, the difference revealing the adenosine‐evoked current. After correction for the liquid junction potential of −18mV, this resulted in an adenosine‐evoked outwardly rectifying current that reversed at −106mV and continued as inward current at more negative membrane potentials (n=5; Fig.4C). To quantify the effect of the membrane potential on the outward current, we measured adenosine‐induced currents at two holding potentials, −70 and −30mV (Fig.4D and G). At a holding potential of −70mV, adenosine evoked an outward current of 13±2pA (n=6; Fig.4D, left panel). The outward current significantly increased to 35±6pA (n=6, P<0.001) at a holding potential of −30mV (Fig.4D, right panel). In A₁ knockout mice, adenosine failed to induce membrane currents (Fig.4E) or resulted in inward currents instead of outward currents, verifying the involvement of A₁ receptors. On average, the adenosine‐mediated effect amounted to −16±4 pA (n=6, P<0.001 as compared to wild‐type) in A₁ knockout mice (Fig.4E and F). To achieve a dose–response curve for the adenosine‐evoked response in wild‐type mice, we tested adenosine concentrations ranging from 3 to 300μm and measured the evoked outward currents (n=9). The results revealed an EC₅₀ value of 22.9μm (Fig.4G). In summary, these experiments indicate that the adenosine‐mediated current is caused by an A₁ receptor‐activated potassium conductance.

Pharmacological characterization of the adenosine‐activated K⁺ conductance

Activation of GIRK mediated by A₁ receptors is a common pathway of adenosinergic neuromodulation (Sickmann & Alzheimer, 2003; Kim & Johnston, 2015). To test the involvement of GIRK in the adenosine‐evoked hyperpolarization of olfactory bulb mitral cells, we applied adenosine in the presence of the GIRK inhibitor Ba²⁺ in current‐clamp experiments using TTX to suppress neuronal activity. Ba²⁺ at 1mm, a concentration sufficient to block GIRK (Sickmann & Alzheimer, 2003; Kim & Johnston, 2015), resulted in depolarization of the membrane of mitral cells and an increase in the frequency of synaptic miniature potentials, which largely recovered following washout of adenosine (Fig.5A). However, Ba²⁺ had no inhibiting effect on the adenosine‐induced hyperpolarization (n=6). In addition, we used canonical blockers of voltage‐gated K⁺ channels at concentrations demonstrated to efficiently and selectively block K⁺ channel subtypes (Gutman etal. 2005; Wei etal. 2005). 4‐AP (10mm; n=7) and TEA (10mm; n=5) did not significantly reduce adenosine‐evoked hyperpolarizations (Fig.5B and C). To reveal the type of K⁺ channel involved, we additionally tested five inhibitors for different K⁺ channel subfamilies for their effect on the adenosine‐evoked hyperpolarization (Fig.5D and E). Only bupivacaine significantly reduced the hyperpolarization. On average, bupivacaine reduced the adenosine‐evoked hyperpolarization by 41.3±9.0% (n=7, P=0.040).

A₁ receptors activate two‐pore domain K⁺ channels

Bupivacaine has been shown to inhibit some of the K2P channels, a large family of background K⁺ channels (Enyedi & Czirjak, 2010; Feliciangeli etal. 2015). To confirm the potential involvement of K2P channels we tested halothane, which activates some of the K2P channels, but inhibits other K2P channels such as bupivacaine‐sensitive THIK channels (Patel etal. 2001; Rajan etal. 2001; Bushell etal. 2002). In voltage‐clamp experiments (V_h −30mV), 3mm halothane evoked a large inward current superimposed by a smaller outward current (Fig.6A), demonstrating that a large part of the background current in mitral cells is carried by K2P channels. The halothane‐evoked inward current presumably is the result of inhibition of halothane‐inhibited K2P channels such as THIK‐1, while the small outward current may reflect halothane‐activated K2P channels such as TREK and TASK (Enyedi & Czirjak, 2010; Feliciangeli etal. 2015). In the presence of halothane, the adenosine‐evoked outward current was significantly inhibited by 33.7±10.4% (n=7, P=0.043; Fig.6B). In addition, bupivacaine reduced the adenosine‐induced outward current by 69.9±8.6% (n=6, P<0.001; Fig.6C). The effect of bupivacaine was partly reversible after 10min of washout of bupivacaine. The results indicate that A₁ receptors modulate a bupivacaine‐sensitive and halothane‐sensitive background K⁺ conductance (Fig.6D).

A₁ receptors fail to modulate synaptic input from sensory axons

Mitral cells receive synaptic input from olfactory sensory neurons. Synaptic transmission between axons of sensory neurons and mitral cells is subject to extensive neuromodulation by GABA and dopamine (Aroniadou‐Anderjaska etal. 2000; Ennis etal. 2001; Maher & Westbrook, 2008). We were interested in whether adenosine was also capable of modulating synaptic input from olfactory sensory neurons in mitral cells. We measured synaptic currents in mitral cells in response to single pulse stimulation of sensory axons (Fig.7A). On average, the amplitude of synaptic currents upon axonal stimulation was −130.7±32.7pA (n=6) and was not affected by application of adenosine (Fig.7B). As a measure of synaptic vesicle fusion in terminals of olfactory sensory axons, we employed a mouse strain in which olfactory sensory neurons express the vesicle fusion marker syn‐pH (Fig.7C). Changes in syn‐pH fluorescence correlate linearly with the transmitter release in receptor axons (Bozza etal. 2004). Fluorescence transients evoked by stimulation of olfactory sensory axons were not reduced in amplitude by application of adenosine (n=4; Fig.7D). The results indicate that the synaptic input from sensory neurons in mitral cells is not influenced by adenosine.

A₁ receptors improve the signal‐to‐noise ratio of mitral cell output signals

Odour information derived from olfactory sensory neurons is processed in the olfactory bulb and propagated by mitral and tufted cells into higher brain centres such as the piriform cortex. The relationship between sensory input signals and mitral cell output signals is one important determinant of odour perception. To assess whether the output signal of mitral cells resulting from sensory stimulation is affected by adenosine, we recorded action currents in the cell‐attached configuration, hence avoiding dialysis of the recorded cell that could compromise excitability. Single stimulation of sensory axons resulted in a biphasic increase in firing rate of the mitral cell (Fig.8A). This biphasic increase in firing rate consisted of an early response of approximately 100ms duration, followed by a late response of approximately 400ms after a short gap devoid of action potential firing (Fig.8A, right panel). The averaged firing rate increased from 5.2±1.6Hz at baseline (averaged from 19s preceding the stimulation) to 29.1±6.0Hz (P<0.001) during the early response phase (averaged from 0 to 100ms after stimulation) and to 10.9±3.1Hz (P<0.001) in the late response phase (averaged from 100 to 500ms after stimulation) (n=10; Fig.8B). The latency of the first action potential after stimulation was 8.4±1.3ms (n=10) and displayed very little variance within a given experiment (Fig.8C), suggesting that the early response resulted from monosynaptic transmission (De Saint Jan etal. 2009) as opposed to disynaptic transmission found upon weak stimulation (Gire etal. 2012). The latency of the first action potential was not altered by adenosine, confirming lack of modulation of the synaptic input from sensory axons (Fig.8D).

We next tested whether adenosine was able to affect the pattern of action potential firing evoked by electrical stimulation of olfactory sensory neurons (Fig.9A). Adenosine reduced the baseline firing rate of mitral cells by 37±6% (n=10, P=0.002), while it had no significant effect on the firing rate in the presence of DPCPX (n=6) (Fig.9B). Adenosine had no effect on the firing rate during the early response in both the absence and the presence of DPCPX, but decreased the firing rate of the late response in wild‐type mice by 36±5% (n=10, P=0.002; Fig.9C and D). As adenosine hyperpolarizes the mitral cell membrane by 2–3mV, we examined whether this hyperpolarization was sufficient to cause the described effect of adenosine on stimulation‐evoked action potential firing. Therefore, we hyperpolarized mitral cells in current‐clamp mode by approximately 3mV by current injection (−15 to −30pA) and recorded action potentials evoked by single pulse stimulation of olfactory sensory neurons (Fig.9E). In current‐clamp recordings, baseline firing frequency was 5.1±1.3Hz (n=5) and increased upon single pulse stimulation to 40.0±6.7Hz (n=5) during the early response and to 16.5±2.0Hz (n=5) during the late response. The induced hyperpolarization decreased the baseline firing rate by 61±5% (n=5, P=0.038) and the late response firing rate by 38±6% (n=5, P=0.018), but had no effect on the early response (Fig.9F–H). Since spontaneous firing reflects the mitral cell output activity independent of specific odour sensation it can be considered as noise, whereas the early response reflects the signal directly evoked by sensory input and propagated to the cortex upon odour sensation as mimicked by stimulation of sensory axons. Hence, the ratio of early response/spontaneous firing is an estimation of the signal‐to‐noise ratio of the mitral cell output. The signal‐to‐noise ratio of 8.4±1.9 under control conditions increased significantly by 62±23% (n=10, P=0.045) upon application of adenosine in the absence but not in the presence of DPCPX (not shown). In addition, the signal‐to‐noise ratio increased by 164±50% (n=5, P=0.002) upon current‐induced hyperpolarization (Fig.9I). We also calculated the signal‐to‐noise ratio of the late response. Since both baseline firing and firing during the late response were reduced by adenosine, the ratio of late response/baseline was not altered by adenosine (Fig.9I). However, it increased upon current‐induced hyperpolarization by 71±27% (n=5; P=0.049). The results indicate that A₁ receptor activation is able to increase the signal‐to‐noise ratio particularly of the firing pattern directly evoked in mitral cells upon synaptic input from sensory neurons and hence to modulate olfactory information that is propagated from the olfactory bulb to other brain areas such as the piriform cortex.

Adenosine modulates potassium channels

Based on membrane conductance measurements and calculation of the reversal potential of the adenosine‐induced ion conductance, we showed that A₁ receptor activation leads to opening of background potassium channels. Our pharmacological data suggest the involvement of K2P channels, which are sensitive to volatile anaesthetics such as chloroform, isoflurane and halothane (Enyedi & Czirjak, 2010; Feliciangeli etal. 2015). Halothane induced a large inward current, suggesting inhibition of K2P channels such as THIK‐1 and THIK‐2, which are highly expressed in the olfactory bulb, including mitral cells (Rajan etal. 2001; Lein etal. 2007), and can form heterodimers (Blin etal. 2014). The halothane‐evoked inward current was superimposed by an outward current, suggesting that in addition to halothane‐inhibited K2P channels, halothane‐activated K2P channels are present in mitral cells, as shown before, for instance, in thalamic neurons (Meuth etal. 2003). Bupivacaine inhibits THIK‐1 (Kang etal. 2014); however, the pharmacology of K2P channels is still poorly understood and the action of bupivacaine alone does not allow for a definite identification of the K2P channel activated by adenosine (Lesage, 2003). For instance, in our experiments barium did not block the adenosine‐evoked hyperpolarization, while in Purkinje neurons a THIK‐1‐like current was reduced by barium (Bushell etal. 2002). Absent barium sensitivity clearly argues against the involvement of GIRK channels, which have been shown to mediate adenosine‐induced hyperpolarization in other types of neurons and are activated by the βγ subunit of G‐proteins coupled to A₁ receptors and other metabotropic receptors (Lüscher & Slesinger, 2010). Several other types of potassium channels have been reported in mitral cells including SK channels, Shaker channels, erg channels and A‐type channels (Fadool etal. 2004; Maher & Westbrook, 2005; Hirdes etal. 2009), but blockers of these channels did not affect the adenosine‐mediated hyperpolarization. Modulation of K2P channels by intracellular signalling pathways in neuronal and non‐neuronal cells has been published (Chen etal. 2006, Comoglio etal. 2014; Leist etal. 2017), but modulation of K2P channels by A₁ receptors has been described only in retinal Müller glial cells so far (Skatchkov etal. 2006). In addition to A₁ receptor expression, we found A_2A receptor expression in mitral cells. In A₁ knockout mice, adenosine application resulted in slight depolarization instead of hyperpolarization, an effect that may be mediated by A_2A receptors, since A_2A receptors often exert opposing effects to A₁ receptors (Cunha etal. 1994; Rebola etal. 2003).

Adenosine modulates mitral cell activity

The pattern of action potentials propagated by mitral cells to cortical structures upon sensory stimulation is one of the main determinants of odour perception (Gire etal. 2013; Uchida etal. 2014). Synaptic input reaching mitral cells via olfactory sensory axons is not affected by adenosine, in contrast to other neuromodulators such as GABA (via GABA_B receptors) and dopamine which reduce neurotransmitter release at olfactory sensory axon terminals (Aroniadou‐Anderjaska etal. 2000; Ennis etal. 2001; Maher & Westbrook, 2008). Firing of mitral cells evoked by synaptic input from olfactory sensory axons comprises two phases, the early response reflecting direct excitation by the olfactory sensory afferents and the delayed response modulated by processing in the bulbar network (Najac etal. 2011). Timing of the early response with respect to the sniffing cycle may determine mitral cell response amplitudes (Smear etal. 2011); however, the latency of the early response to olfactory nerve stimulation is unaltered by adenosine, suggesting that adenosine does not impact temporal odour coding by early response modulation. In contrast, the frequency of spontaneous firing, but also of the firing in the late phase of the response, was reduced. The latter might result from A₁ receptor‐sensitive integration of feedback excitation and inhibition (Hayar etal. 2004; Giridhar etal. 2011; Gire etal. 2012; Carey etal. 2015; Liu etal. 2015; Parsa etal. 2015) and the results suggest that the temporal pattern of glomerular network activity is shaped by A₁ receptors. Since the temporal pattern of glomerular network activity is decisive for odour perception (Shusterman etal. 2011; Haddad etal. 2013; Tripathy etal. 2013; Nunez‐Parra etal. 2014; Uchida etal. 2014), A₁ receptors might thus fine‐tune odour perception. Recently it was reported that the input–output transformation in mitral and tufted cells contributes to the perception of odour concentration invariances (Storace & Cohen, 2017). Tuning of the input–output transformation by neuromodulators such as adenosine might therefore adjust the perception of odour concentration invariances. An increase in signal‐to‐noise ratio of the input–output transformation of mitral cells, comparable to the one described in the present study, has been found by stimulation of noradrenalin receptors in the olfactory bulb, which results in improved odour detection thresholds in behavioural tests (Escanilla etal. 2010; Manella etal. 2017). Adenosine increases the signal‐to‐noise ratio of the evoked firing pattern in mitral cells that are excited by the sensory stimulus; odour‐evoked mitral cell responses, however, consist of a complex ensemble of both excited and inhibited mitral cells (Economo etal. 2016). In contrast to excitatory mitral cell responses, the signal‐to‐noise ratio of inhibitory responses would be attenuated by an adenosine‐mediated reduction of spontaneous firing (noise), suggesting that the action of adenosine on information processing in the olfactory bulb might be even more complex than indicated by the present study. It must be noted that in awake animals, the response pattern of action potential firing upon sensory input, but also the spontaneous action potential firing of mitral cells, can differ from action potential firing in anaesthetized animals or slice preparations. Rinberg etal. (2006) reported an increased spontaneous firing rate of mitral cells in the awake situation compared to anaesthetized mice, while Kollo etal. (2014) found that about one‐third of mitral cells were silent in awake mice, but not in anaesthetized mice. Since K2P channels are affected by anaesthetics such as halothane and isofluorane used in some of the studies using anaesthetized animals, activation and/or inhibition of K2P channels in mitral cells by these anaesthetics may partly account for the differences between awake and anaesthetized mice.

Purinergic signalling has been described as affecting olfactory bulb neurons and glial cells via P2Y receptors and adenosine receptors, but the role of adenosine in odour information processing has not been elucidated before (Rieger etal. 2007; Doengi etal. 2008; Fischer etal. 2012; Roux etal. 2015). It should be noted that in the study by Roux etal. (2015) A₁ receptors appeared to increase excitability of mitral cells, whereas in our study activation of A₁ receptors reduced excitability, suggesting that adenosinergic modulation of mitral cell activity is intricate and variable.

In conclusion, our study shows that adenosine is able to modulate mitral cell membrane properties, attenuate network activity and tune the input–output relationship of mitral cell signalling. Hence, purinergic signalling might significantly contribute to odour information processing in the olfactory bulb.

Competing interest

The authors declare no conflict of interest.

Author contributions

Design of the work: C.L., D.H. and N.R. Acquisition and analysis of data, providing material: C.L., D.H., J.B., K.S., M.B., M.C. N.B., N.R., S.W. and T.F. Writing the manuscript: C.L., D.H., M.C. and N.R. All authors revised and approved the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

Financial support by the Deutsche Forschungsgemeinschaft (LO 779/6‐1 and HI 1288/3‐1), the Landesforschungsförderung Hamburg (Read Me) and the Swedish Research Council (Dnr: 2016‐01381) is gratefully acknowledged.

•

Natalie Rotermund received her PhD in Neurophysiology from the University of Hamburg. After working in the medical device industry for some years, she decided to return to university and follow her interest in fundamental research. She is currently employed as postdoctoral researcher in the Division of Neurophysiology at the University of Hamburg. Her research focuses on the understanding of purinergic signalling in the central nervous system, with a specialization in electrophysiological and imaging techniques.

• Aroniadou‐Anderjaska V, Zhou FM, Priest CA, Ennis M & Shipley MT (2000). Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA_B heteroreceptors. J Neurophysiol84, 1194–1203. [PubMed] [Google Scholar]
• Blin S, Chatelain FC, Feliciangeli S, Kang D, Lesage F & Bichet D (2014). Tandem pore domain halothane‐inhibited K⁺ channel subunits THIK1 and THIK2 assemble and form active channels. J Biol Chem289, 28202–28212. [PMC free article] [PubMed] [Google Scholar]
• Boison D (2009). Adenosine‐based modulation of brain activity. Curr Neuropharmacol7, 158–159. [PMC free article] [PubMed] [Google Scholar]
• Bozza T, McGann JP, Mombaerts P & Wachowiak M (2004). In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron42, 9–21. [PubMed] [Google Scholar]
• Bushell T, Clarke C, Mathie A & Robertson B (2002). Pharmacological characterization of a non‐inactivating outward current observed in mouse cerebellar Purkinje neurones. Br J Pharmacol135, 705–712. [PMC free article] [PubMed] [Google Scholar]
• Carey RM, Sherwood WE, Shipley MT, Borisyuk A & Wachowiak M (2015). Role of intraglomerular circuits in shaping temporally structured responses to naturalistic inhalation‐driven sensory input to the olfactory bulb. JNeurophysiol113, 3112–3129. [PMC free article] [PubMed] [Google Scholar]
• Chen X, Talley EM, Patel N, Gomis A, McIntire WE, Dong B, Viana F, Garrison JC & Bayliss DA (2006). Inhibition of a background potassium channel by Gq protein α‐subunits. Proc Natl Acad Sci USA103, 3422–3427. [PMC free article] [PubMed] [Google Scholar]
• Clark BD, Kurth‐Nelson ZL & Newman EA (2009). Adenosine‐evoked hyperpolarization of retinal ganglion cells is mediated by G‐protein‐coupled inwardly rectifying K⁺ and small conductance Ca²⁺‐activated K⁺ channel activation. J Neurosci29, 11237–11245. [PMC free article] [PubMed] [Google Scholar]
• Comoglio Y, Levitz J, Kienzler MA, Lesage F, Isacoff EY & Sandoz G (2014). Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid. Proc Natl Acad Sci USA111, 13547–13552. [PMC free article] [PubMed] [Google Scholar]
• Cunha RA (2005). Neuroprotection by adenosine in the brain: From A₁ receptor activation to A_2A receptor blockade. Purinergic Signal1, 111–134. [PMC free article] [PubMed] [Google Scholar]
• Cunha RA, Johansson B, van der Ploeg I, Sebastião AM, Ribeiro JA & Fredholm BB (1994). Evidence for functionally important adenosine A_2a receptors in the rat hippocampus. Brain Res649, 208–216. [PubMed] [Google Scholar]
• De Saint Jan D, Hirnet D, Westbrook GL & Charpak S (2009). External tufted cells drive the output of olfactory bulb glomeruli. J Neurosci29, 2043–2052. [PMC free article] [PubMed] [Google Scholar]
• Dias RB, Rombo DM, Ribeiro JA, Henley JM & Sebastiao AM (2013). Adenosine: setting the stage for plasticity. Trends Neurosci36, 248–257. [PubMed] [Google Scholar]
• Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ & Freeman TC (1996). Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol118, 1461–1468. [PMC free article] [PubMed] [Google Scholar]
• Doengi M, Deitmer JW & Lohr C (2008). New evidence for purinergic signaling in the olfactory bulb: A_2A and P2Y₁ receptors mediate intracellular calcium release in astrocytes. FASEB J22, 2368–2378. [PubMed] [Google Scholar]
• Dos Santos‐Rodrigues A, Pereira MR, Brito R, de Oliveira NA & Paes‐de‐Carvalho R (2015). Adenosine transporters and receptors: key elements for retinal function and neuroprotection. Vitam Horm98, 487–523. [PubMed] [Google Scholar]
• Economo MN, Hansen KR & Wachowiak M (2016). Control of mitral/tufted cell output by selective inhibition among olfactory bulb glomeruli. Neuron20, 397–411. [PMC free article] [PubMed] [Google Scholar]
• Ennis M, Zhou F, Ciombor KJ, Aroniadou‐Anderjaska V, Hayar A & Borrelli E (2001). Dopamine D2 receptor‐mediated presynaptic inhibition of olfactory nerve terminals. J Neurophysiol86, 2986–2997. [PubMed] [Google Scholar]
• Enyedi P & Czirjak G (2010). Molecular background of leak K⁺ currents: two‐pore domain potassium channels. Physiol Rev90, 559–605. [PubMed] [Google Scholar]
• Escanilla O, Arrellanos A, Karnow A, Ennis M & Linster C (2010). Noradrenergic modulation of behavioral odor detection and discrimination thresholds in the olfactory bulb. Eur J Neurosci32, 458–468. [PubMed] [Google Scholar]
• Fadool DA, Tucker K, Perkins R, Fasciani G, Thompson RN, Parsons AD, Overton JM, Koni PA, Flavell RA & Kaczmarek LK (2004). Kv1.3 channel gene‐targeted deletion produces “Super‐Smeller Mice” with altered glomeruli, interacting scaffolding proteins, and biophysics. Neuron41, 389–404. [PMC free article] [PubMed] [Google Scholar]
• Feliciangeli S, Chatelain FC, Bichet D, Lesage F (2015). The family of K2P channels: salient structural and functional properties. J Physiol593, 2587–2603. [PMC free article] [PubMed] [Google Scholar]
• Fischer T, Rotermund N, Lohr C & Hirnet D (2012). P2Y₁ receptor activation by photolysis of caged ATP enhances neuronal network activity in the developing olfactory bulb. Purinergic Signal8, 191–198. [PMC free article] [PubMed] [Google Scholar]
• Fredholm BB, Chen JF, Cunha RA, Svenningsson P & Vaugeois JM (2005). Adenosine and brain function. Int Rev Neurobiol63, 191–270. [PubMed] [Google Scholar]
• Gire DH, Franks KM, Zak JD, Tanaka KF, Whitesell JD, Mulligan AA, Hen R & Schoppa NE (2012). Mitral cells in the olfactory bulb are mainly excited through a multistep signaling path. J Neurosci32, 2964–2975. [PMC free article] [PubMed] [Google Scholar]
• Gire DH, Restrepo D, Sejnowski TJ, Greer C, De Carlos JA & Lopez‐Mascaraque L (2013). Temporal processing in the olfactory system: can we see a smell? Neuron78, 416–432. [PMC free article] [PubMed] [Google Scholar]
• Giridhar S, Doiron B & Urban NN (2011). Timescale‐dependent shaping of correlation by olfactory bulb lateral inhibition. Proc Natl Acad Sci USA108, 5843–5848. [PMC free article] [PubMed] [Google Scholar]
• Grundy D (2015). Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology. J Physiol593, 2547–2549. [PMC free article] [PubMed] [Google Scholar]
• Gundlfinger A, Bischofberger J, Johenning FW, Torvinen M, Schmitz D & Breustedt J (2007). Adenosine modulates transmission at the hippocampal mossy fibre synapse via direct inhibition of presynaptic calcium channels. J Physiol582, 263–277. [PMC free article] [PubMed] [Google Scholar]
• Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, Pardo LA, Robertson GA, Rudy B, Sanguinetti MC, Stühmer W & Wang X (2005). International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage‐gated potassium channels. Pharmacol Rev57, 473–508. [PubMed] [Google Scholar]
• Haddad R, Lanjuin A, Madisen L, Zeng H, Murthy VN & Uchida N (2013). Olfactory cortical neurons read out a relative time code in the olfactory bulb. Nat Neurosci16, 949–957. [PMC free article] [PubMed] [Google Scholar]
• Hayar A, Karnup S, Ennis M & Shipley MT (2004). External tufted cells: a major excitatory element that coordinates glomerular activity. J Neurosci24, 6676–6685. [PMC free article] [PubMed] [Google Scholar]
• Hirdes W, Napp N, Wulfsen I, Schweizer M, Schwarz JR & Bauer CK (2009). Erg K⁺ currents modulate excitability in mouse mitral/tufted neurons. Pflugers Arch459, 55–70. [PubMed] [Google Scholar]
• Housley GD, Bringmann A & Reichenbach A (2009). Purinergic signaling in special senses. Trends Neurosci32, 128–141. [PubMed] [Google Scholar]
• Johansson B, Georgiev V & Fredholm BB (1997). Distribution and postnatal ontogeny of adenosine A_2A receptors in rat brain: comparison with dopamine receptors. Neuroscience80, 1187–1207. [PubMed] [Google Scholar]
• Johansson B, Halldner L, Dunwiddie TV, Masino SA, Poelchen W, Giménez‐Llort L, Escorihuela RM, Fernández‐Teruel A, Wiesenfeld‐Hallin Z, Xu XJ, Hårdemark A, Betsholtz C, Herlenius E & Fredholm BB (2001). Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A₁ receptor. Proc Natl Acad Sci USA98, 9407–9412. [PMC free article] [PubMed] [Google Scholar]
• Kaelin‐Lang A, Lauterburg T & Burgunder JM (1999). Expression of adenosine A_2a receptors gene in the olfactory bulb and spinal cord of rat and mouse. Neurosci Lett261, 189–191. [PubMed] [Google Scholar]
• Kang D, Hogan JO & Kim D (2014). THIK‐1 (K2P13.1) is a small‐conductance background K⁺ channel in rat trigeminal ganglion neurons. Pflugers Arch466, 1289–1300. [PMC free article] [PubMed] [Google Scholar]
• Kim CS & Johnston D (2015). A₁ adenosine receptor‐mediated GIRK channels contribute to the resting conductance of CA1 neurons in the dorsal hippocampus. J Neurophysiol113, 2511–2523. [PMC free article] [PubMed] [Google Scholar]
• Kollo M, Schmaltz A, Abdelhamid M, f*ckunaga I & Schaefer AT (2014). ‘Silent’ mitral cells dominate odor responses in the olfactory bulb of awake mice. Nat Neurosci17, 1313–1315. [PMC free article] [PubMed] [Google Scholar]
• Langer D, Hammer K, Koszalka P, Schrader J, Robson S & Zimmermann H (2008). Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res334, 199–217. [PubMed] [Google Scholar]
• Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA & Jones AR (2007). Genome‐wide atlas of gene expression in the adult mouse brain. Nature445, 168–176. [PubMed] [Google Scholar]
• Leist M, Rinné S, Datunashvili M, Aissaoui A, Pape HC, Decher N, Meuth SG & Budde T (2017). Acetylcholine‐dependent upregulation of TASK‐1 channels in thalamic interneurons by a smooth muscle‐like signalling pathway. J Physiol595, 5875–5893. [PMC free article] [PubMed] [Google Scholar]
• Lesage F (2003). Pharmacology of neuronal background potassium channels. Neuropharmacology44, 1–7. [PubMed] [Google Scholar]
• Liu S, Shao Z, Puche A, Wachowiak M, Rothermel M & Shipley MT (2015). Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output. J Neurosci35, 5680–5692. [PMC free article] [PubMed] [Google Scholar]
• Lohr C, Grosche A, Reichenbach A & Hirnet D (2014). Purinergic neuron‐glia interactions in sensory systems. Pflugers Arch466, 1859–1872. [PubMed] [Google Scholar]
• Lopes LV, Cunha RA, Kull B, Fredholm BB & Ribeiro JA (2002). Adenosine A_2A receptor facilitation of hippocampal synaptic transmission is dependent on tonic A₁ receptor inhibition. Neuroscience112, 319–329. [PubMed] [Google Scholar]
• Lüscher C & Slesinger PA (2010). Emerging roles for G protein‐gated inwardly rectifying potassium (GIRK) channels in health and disease. Nat Rev Neurosci11, 301–315. [PMC free article] [PubMed] [Google Scholar]
• Mahan LC, McVittie LD, Smyk‐Randall EM, Nakata H, Monsma FJ Jr, Gerfen CR & Sibley DR. (1991). Cloning and expression of an A₁ adenosine receptor from rat brain. Mol Pharmacol40, 1–7. [PubMed] [Google Scholar]
• Maher BJ & Westbrook GL (2005). SK channel regulation of dendritic excitability and dendrodendritic inhibition in the olfactory bulb. J Neurophysiol94, 3743–3750. [PubMed] [Google Scholar]
• Maher BJ & Westbrook GL (2008). Co‐transmission of dopamine and GABA in periglomerular cells. J Neurophysiol99, 1559–1564. [PubMed] [Google Scholar]
• Manella LC, Petersen N & Linster C (2017). Stimulation of the locus coeruleus modulates signal‐to‐noise ratio in the olfactory bulb. J Neurosci37, 11605–11615. [PMC free article] [PubMed] [Google Scholar]
• Meuth SG, Budde T, Kanyshkova T, Broicher T, Munsch T & Pape HC (2003). Contribution of TWIK‐related acid‐sensitive K⁺ channel 1 (TASK1) and TASK3 channels to the control of activity modes in thalamocortical neurons. JNeurosci23, 6460–6469. [PMC free article] [PubMed] [Google Scholar]
• Najac M, De Saint Jan D, Reguero L, Grandes P & Charpak S (2011). Monosynaptic and polysynaptic feed‐forward inputs to mitral cells from olfactory sensory neurons. J Neurosci31, 8722–8729. [PMC free article] [PubMed] [Google Scholar]
• Nunez‐Parra A, Li A & Restrepo D (2014). Coding odor identity and odor value in awake rodents. Prog Brain Res208, 205–222. [PMC free article] [PubMed] [Google Scholar]
• Parsa PV, D'Souza RD & Vijayaraghavan S (2015). Signaling between periglomerular cells reveals a bimodal role for GABA in modulating glomerular microcircuitry in the olfactory bulb. Proc Natl Acad Sci USA112, 9478–9483. [PMC free article] [PubMed] [Google Scholar]
• Patel AJ, Honoré E, Lesage F, Fink M, Romey G & Lazdunski M (2001). Inhalational anesthetics activate two‐pore‐domain background K⁺ channels. Nat Neurosci2, 422–436. [PubMed] [Google Scholar]
• Rajan S, Wischmeyer E, Karschin C, Preisig‐Muller R, Grzeschik KH, Daut J, Karschin A & Derst C (2001). THIK‐1 and THIK‐2, a novel subfamily of tandem pore domain K⁺ channels. J Biol Chem276, 7302–7311. [PubMed] [Google Scholar]
• Rebola N, Sebastião AM, de Mendonca A, Oliveira CR, Ribeiro JA & Cunha RA (2003). Enhanced adenosine A_2A receptor facilitation of synaptic transmission in the hippocampus of aged rats. J Neurophysiol90, 1295–1303. [PubMed] [Google Scholar]
• Reppert SM, Weaver DR, Stehle JH & Rivkees SA (1991). Molecular cloning and characterization of a rat A1‐adenosine receptor that is widely expressed in brain and spinal cord. Mol Endocrinol5, 1037–1048. [PubMed] [Google Scholar]
• Ribeiro JA & Sebastiao AM (2010). Modulation and metamodulation of synapses by adenosine. Acta Physiol (Oxf)199, 161–169. [PubMed] [Google Scholar]
• Rieger A, Deitmer JW & Lohr C (2007). Axon‐glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulb. Glia55, 352–359. [PubMed] [Google Scholar]
• Rinberg D, Koulakov A & Gelperin A (2006). Sparse odor coding in awake behaving mice. J Neurosci26, 8857–8865. [PMC free article] [PubMed] [Google Scholar]
• Rosin DL, Robeva A, Woodard RL, Guyenet PG & Linden J (1998). Immunohistochemical localization of adenosine A_2A receptors in the rat central nervous system. J Comp Neurol401, 163–186. [PubMed] [Google Scholar]
• Roux L, Madar A, Lacroix MM, Yi C, Benchenane K & Giaume C (2015). Astroglial connexin 43 hemichannels modulate olfactory bulb slow oscillations. J Neurosci35, 15339–15352. [PMC free article] [PubMed] [Google Scholar]
• Shusterman R, Smear MC, Koulakov AA & Rinberg D (2011). Precise olfactory responses tile the sniff cycle. Nat Neurosci14, 1039–1044. [PubMed] [Google Scholar]
• Sickmann T & Alzheimer C (2003). Short‐term desensitization of G‐protein‐activated, inwardly rectifying K⁺ (GIRK) currents in pyramidal neurons of rat neocortex. JNeurophysiol90, 2494–2503. [PubMed] [Google Scholar]
• Skatchkov SN, Eaton MJ, Shuba YM, Kucheryavykh YV, Derst C, Veh RW, Wurm A, Iandiev I, Pannicke T, Bringmann A & Reichenbach A (2006). Tandem‐pore domain potassium channels are functionally expressed in retinal (Müller) glial cells. Glia53, 266–276. [PubMed] [Google Scholar]
• Smear M, Shusterman R, O'Connor R, Bozza T & Rinberg D (2011). Perception of sniff phase in mouse olfaction. Nature479, 397–400. [PubMed] [Google Scholar]
• Stavermann M, Meuth P, Doengi M, Thyssen A, Deitmer JW & Lohr C (2015). Calcium‐induced calcium release and gap junctions mediate large‐scale calcium waves in olfactory ensheathing cells in situ. Cell Calcium58, 215–225. [PubMed] [Google Scholar]
• Storace DA & Cohen LB (2017). Measuring the olfactory bulb input‐output transformation reveals a contribution to the perception of odorant concentration invariance. Nat Commun8, 81. [PMC free article] [PubMed] [Google Scholar]
• Svenningsson P, Le Moine C, Kull B, Sunahara R, Bloch B & Fredholm BB (1997). Cellular expression of adenosine A2A receptor messenger RNA in the rat central nervous system with special reference to dopamine innervated areas. Neuroscience80, 1171–1185. [PubMed] [Google Scholar]
• Thyssen A, Hirnet D, Wolburg H, Schmalzig G, Deitmer JW & Lohr C (2010). Ectopic vesicular neurotransmitter release along sensory axons mediates neurovascular coupling via glial calcium signaling. Proc Natl Acad Sci USA107, 15258–15263. [PMC free article] [PubMed] [Google Scholar]
• Tripathy SJ, Padmanabhan K, Gerkin RC & Urban NN (2013). Intermediate intrinsic diversity enhances neural population coding. Proc Natl Acad Sci USA110, 8248–8253. [PMC free article] [PubMed] [Google Scholar]
• Uchida N, Poo C & Haddad R (2014). Coding and transformations in the olfactory system. Annu Rev Neurosci37, 363–385. [PubMed] [Google Scholar]
• Wei AD, Gutman GA, Aldrich R, Chandy KG, Grissmer S & Wulff H (2005). International Union of Pharmacology. LII. Nomenclature and molecular relationships of calcium‐activated potassium channels. Pharmacol Rev57, 463–472. [PubMed] [Google Scholar]