Tonic inhibition sets the state of excitability in olfactory bulb granule cells

J Physiol 591.7 (2013) pp 1841–1850 1841

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Tonic inhibition sets the state of excitability in olfactorybulb granule cells

Christina Labarrera1,2, Michael London2 and Kamilla Angelo1

1Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark2Department of Neurobiology, The Hebrew University of Jerusalem, Jerusalem, Israel

Key points

• Granule cells are the main source of inhibition in the olfactory bulb (i.e. the first station ofodour processing in the mammalian brain), but very little is known about the inhibition thatacts upon them.

• Using in vivo whole cell patch clamp recordings in anaesthetized mice we report the followingnew findings:

• We found odour-evoked responses to be rare (seen only in 18% of the odour presentations,and only in cells that showed also evoked excitatory responses to odours).

• We report for the first time the presence of tonic inhibition in the olfactory bulb.• We show that tonic inhibition dominates over phasic synaptic inhibition evoked by odours,

thereby being the key regulator shaping the granule cells spike output.• Preliminary (in vivo) evidence suggests that sensory evoked phasic inhibition onto granule

cells is provided by deep short axon cells in the olfactory bulb.

Abstract GABAergic granule cells (GCs) regulate, via mitral cells, the final output fromthe olfactory bulb to piriform cortex and are central for the speed and accuracy of odourdiscrimination. However, little is known about the local circuits in which GCs are embeddedand how GCs respond during functional network activity. We recorded inhibitory and excitatorycurrents evoked during a single sniff-like odour presentation in GCs in vivo. We found thatsynaptic excitation was extensively activated across cells, whereas phasic inhibition was rare.Furthermore, our analysis indicates that GCs are innervated by a persistent firing of deep shortaxon cells that mediated the inhibitory evoked responses. Blockade of GABAergic synaptic inputonto GCs revealed a tonic inhibitory current mediated by furosemide-sensitive GABAA receptors.The average current associated with this tonic GABAergic conductance was 3-fold larger thanthat of phasic inhibitory postsynaptic currents. We show that the pharmacological blockageof tonic inhibition markedly increased the occurrence of supra-threshold responses during anodour-stimulated sniff. Our findings suggest that GCs mediate recurrent or lateral inhibition,depending on the ambient level of extracellular GABA.

(Received 27 July 2012; accepted after revision 10 January 2013; first published online 14 January 2013)Corresponding author C. Labarrera: The Silberman building 3th floor, room 343, Givat Ram, Neurobiology, TheHebrew University of Jerusalem, 91904 Jerusalem, Israel. Email: [email protected]

Abbreviations AP, action potential; dSACs, deep short axon cells; EPSCs, evoked excitatory postsynaptic currents;GCs, granule cells; IPSCs, inhibitory postsynaptic currents.

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society DOI: 10.1113/jphysiol.2012.241851

1842 C. Labarrera and others J Physiol 591.7

Introduction

Inhibitory interneurons are integral elements of localmicrocircuits. In the olfactory bulb, the output of theprincipal neurons is gated by recurrent and lateralinhibition mediated by GABAergic granule cells (GCs)located in the innermost layer of the bulb. GCs have longbeen known to be excited by mitral cells and in turn inhibitmitral cells. These synaptic interactions are mediated bybidirectional dendro-dendritic synapses between GCs andmitral cell lateral dendrites. Calcium influx shapes trans-mitter release from GCs, and is distributed and regulatedby complex regenerative response properties (Egger et al.2003, Pinato & Midtgaard, 2005). The relationshipbetween synaptic input, regenerative responses andGABA release remains unclear, but action potentials(APs) are not an absolute requirement of synapticrelease (Jahr & Nicoll, 1980, Isaacson & Strowbridge,1998).

Indirect evidence suggests that olfactory GCs areunder some form of suppressive inhibitory drive. In vivostudies have shown that although GCs receive barragesof excitatory postsynaptic potentials, they rarely spike.Additionally, with prolonged odour stimulation, GCexcitation adapts quickly between the first and secondrespiratory cycle (Cang & Isaacson, 2003, Margrie &Schaefer, 2003). Slice studies demonstrate that GCsare synaptically coupled to deep short axon cells(dSACs) in the GC layer (Pressler & Strowbridge, 2006,Eyre et al. 2008). How GCs are controlled by theseinhibitory connections or whether they are controlledthrough alternative inhibitory mechanisms during odourprocessing remains unknown.

Recently, the synaptic activity of GCs has unequivocallybeen linked to neuronal processing time in odourdiscrimination in mice (Abraham et al. 2010). Odourrecognition occurs within the time frame of a singlerespiratory cycle (<300 ms) in a manner that involvesGC lateral circuits (Uchida & Mainen, 2003, Koulakovet al. 2005). However, the synaptic input to GCsand their responses during a respiratory cycle remainunexplored.

The present study investigates the synaptic inputand spiking behaviour of GCs during a 100 msinhalation-aligned odour presentation in vivo. Ourrecordings of odour-evoked excitatory and inhibitorycurrents showed that GCs are predominantly excited bythis sniff-like olfactory stimulus, whereas spiking is curbedby tonic rather than phasic inhibition. We demonstratedthat the level of tonic conductance was 3-fold higherthan the level of phasic synaptic inhibition and thatblockage of tonic inhibition changed the GC input–outputrelationship.

Methods

In vivo electrophysiology

The animal procedures were conducted in accordancewith the European Union’s Council Directive 86/609/EEC.In vivo whole-cell recordings were performed as describedby Margrie & Schaefer (2003). Female C57BL6/J mice(postnatal day 25–30; Taconic, Bomholtvej 10, 8680 Ry,Denmark) were anaesthetized with ketamine/xylazine(100/10 mg kg−1). The selection criteria for GCs were:recording depth >340 μm (434 ± 66 μm, n = 62), cellularcapacitance <12 pF (8.6 ± 2.3 pF, n = 53) and inputresistance >200 M� (Rin,normalinternal = 384 ± 130 M�,n = 21; Rin,cesiuminternal = 1137 ± 638 M�, n = 50). All ofthe recordings were obtained using a Multiclamp 700Bamplifier (Mollecular Devices, CA). The signal was filteredat 2 kHz It was digitized using ITC-18 (HEKA GmbH,Germany) and analysed using Neuromatics/NClampsoftware (http://www.neuromatic.thinkrandom.com).The pipette solution for current-clamp recordingscontained 130 mM methanesulfonic acid, 10 mM Hepes,7 mM KCl, 0.05 mM EGTA in KOH, 2 mM Na2ATP, 2 mM

MgATP and 0.5 mM Na2GTP, pH 7.2, with KOH. Acaesium-based solution was used for all voltage-clamprecordings: 140 mM methanesulfonic acid, 4 mM Tea-Cl,2–5 mM QX-314, 15 mM Hepes, 4 mM NaCl, 1 mM EGTAin CsOH, 2 mM Na2ATP, 2 mM MgATP and 0.5 mM

Na2GTP, pH 7.2, with CsOH. Furosemide and Gabazine(SR-95531) (www.sigmaaldrich.com) were each applieddirectly on the craniotomy in doses of 10–20 μl of 0.5 mM.Animals were terminated via cervical dislocation underketamine/xylazine anaesthesia.

Odour stimulation

Respiration was monitored using a thermocouple placedin a 1 × 1 mm hole that accessed the nasal cavity. Theslope of the respiratory signal just prior to inhalation wasdetected with a custom made window discriminator, anda Transitor-Transitor Logic (TTL) signal was utilized toopen a final valve that controlled the flow of odorousair presented to the left nostril. To mimic naturalsniffing, the odours were presented as individual shortpulses that lasted 100 ms at the beginning of inhalation(Fig. 1A, inset). Odours (1:10 in mineral oil: isoamylacetate (AA), ethyl butyrate (EB), nonanoic acid (NA),and a combination of cineole, lavender, carvone andbenzaldehyde (Mix)) were presented with the inhalationusing a custom-built olfactometer (Bodyak & Slotnick,1999).

Data analysis

The data were analysed using Neuromatics.v2/IgorPro.v5and Excel software. A subthreshold odour-evoked

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society

J Physiol 591.7 Tonic inhibition modulates GC spiking 1843

response was analysed on the average trace from fivetrials and measured as the voltage difference (�V )between baseline and peak amplitude. Odour pre-sentations with a �V stimulated cycle/�V spontaneous cycle ratio<1 were categorized as a non-response. Spikelets weredetected at a threshold of 7 mV (Pinato & Midtgaard,2005) using the threshold-above-baseline event detectionfunction in Neuromatic.v2, and further characterizedas dV /dt > 0.6 V s−1 (Zelles et al. 2006). The sameapproach was used to detect spontaneous and evokedexcitatory postsynaptic currents (EPSCs) and inhibitorypostsynaptic currents (IPSCs) using a threshold of 3–7 pA,depending on the peak-to-peak noise level (Figs 2B and3A and B). The criteria of an evoked average odourresponse from our voltage-clamp recordings were setat an average peak of 5 pA above baseline for bothexcitatory and inhibitory inputs. Prior to averaging, theindividually stimulated trials were aligned to the time ofthe peak of the stimulated respiration cycle. The time lagbetween inhibition and excitation was calculated usingtwo methods. First, we calculated the time where theabsolute value of the trace (IPSC or EPSC) reaches 10%of peak amplitude. In the second method we calculatedthe derivative of the current trace with respect to timefor both inhibition and excitation. The delay betweenthe two traces was calculated as the time differencebetween the time of the minimum derivatives of theexcitation and the maximum derivative of the inhibitionwithin a time window of 50 ms from onset. Actionpotential threshold was detected at 10 mV ms−1 on aphase plot (dV /dt vs. V ; Naundorf et al. 2006). Statisticalsignificance (P < 0.05) was assessed using a two-tailedStudent’s t test, and all data are reported as mean ± SDunless otherwise specified. Classification of recordingsas originating from dSACs was based on a criterionof persistent spiking upon current injection or odourstimulation.

Results

Spiking activity in GCs upon odour presentation

In vivo whole-cell patch-clamp recordings were performedfrom inhibitory GCs in the olfactory bulb in anaesthetizedmice presented with a 100 ms inhalation-aligned odourpulse (Fig. 1A). Despite the central role of GCs incontrolling the final output from the bulb, theirspontaneous spike rate in vivo is low (<0.4 Hz, n = 20)(Cang & Isaacson, 2003, Margrie & Schaefer, 2003).In our experiments, full somatic spikes were recordedin 22% of the odour presentations, while subthresholddepolarization was the most widespread response typewith 58% (207 of 355; spontaneous vs. stimulated;peak depolarization: –55.8 ± 6.3 vs. –64.1 ± 6.8 mV,respectively, P < 0.001; �V m: 10.6 ± 4.9 vs. 4.4 ± 3.0 mV,

respectively, P < 0.001; area: 2376 ± 1273 vs. 23 ± 118 mVms, respectively, P < 0.001; n = 15 cells; Fig. 1A). Thesuprathreshold responses observed were in the form offull-amplitude APs (77 of 355 presentations; Fig. 1A, blue)or spikelets (50 of 355 presentations; Fig. 1A, black),21 out of 355 presentations did not evoke a response;Fig. 1C. The spikelets were characterized by a fast riseand small amplitude and often occurred in bursts ata frequency of ∼15 Hz (Rise time10−90%: 1.2 ± 0.19 ms;Amp: 10.5 ± 0.2 mV; dV /dt > 0.6).

Evoked responses in GCs were not selective to thespecific type of odour (Fig. 1C, bottom). Rather, theresponse form, evoked by one odour, was also the mostlikely type of response to the additional odours presented,suggesting that GCs have a preferred response profile(Fig. 1B).

GC excitatory and inhibitory synaptic currentsin response to odour stimulation

Rare and delayed spiking in GCs is partially determinedby their intrinsic properties (Schoppa & Westbrook, 1999,Kapoor & Urban, 2006) but may also reflect synapticinhibition. Previous studies showed that it is possibleto successfully voltage-clamp olfactory GCs (Schoppa &Westbrook, 1999, Balu et al. 2007). Thus, we appliedthe reversal potential clamp technique to isolate andseparate odour-evoked inhibitory and excitatory synapticresponses in GCs. To reduce distortion of synapticcurrents due to space-clamp (Williams & Mitchell, 2008,Poleg-Polsky & Diamond, 2011), we used a caesium-basedinternal solution that contained ion channel blockers topharmacologically minimize the active intrinsic propertiesof the dendrites (see Methods). The excitatory synapticinput evoked by sniff stimulation was recorded at–75 mV, whereas the inhibitory currents were monitoredat approximately +20 mV. A stimulus-related EPSC burstwas observed in response to most of the presentations(135 of 189 presentations; Fig. 2 Btop and C). In contrast,a low occurrence of inhibitory evoked responses wasobserved (35 of 189 presentations; Fig. 2 BBottom andC). In rare cases of evoked IPSCs, the currents werebroadly activated and only observed in GCs with evokedexcitation. Comparisons of the timing of the excitatoryand inhibitory currents revealed a delay of inhibitionrelative to excitation (see Methods; analysed at 10% of thepeak amplitude and using maximum derivatives: IPSCtime lag (mean ± SEM), 19.9 ± 5.17 and 11.3 ± 6.4 ms,respectively, N = 10 cells, n = 24 stimulations; Fig. 2D,lower panel).

Persistently firing interneurons mediate rare synapticinhibition of GCs

While performing blind in vivo patch-clamping at adepth of >400 μm we occasionally encountered neurons



with AP characteristics which clearly differed fromGCs. We suspect these cells to be dSACs (GCs vs.dSACs; APthres: –39.2 ± 6.2 vs. –46.6 ± 3.7 mV, P < 0.01;APamp: 31.0 ± 6 vs. 38.1 ± 10.5 mV, P < 0.05; n = 30random spikes, data not shown). In these suspecteddSACs the firing probability in response to odour pre-

sentation was high and the AP number during a briefdepolarization was high compared with GCs (dSACs vs.GCs; APPo = 0.90 vs. 0.22; AP number per cycle: 3.8 ± 1.8vs. 2.87 ± 2.3, P < 0.05; n = 45 random stimulationsfrom each group). Most importantly these interneuronsexhibited long-lasting spiking activity upon current

Figure 1. Odour-evoked responses of olfactory bulb GCs in vivoA, representative traces of the three observed output types in response to inhalation-aligned 100 ms odourpresentation (red bar): subthreshold (grey), spikelets (black) and action potential (blue). Inset, odour deliveryoccurred during the square pulse, and the black sinusoidal is the respiration signal. Above, magnifications of eachresponse, i.e. subthreshold, spikelets and an action potential. B, raster plots of three individual GCs presented withfour different odour stimuli. The vertical grey bar represents the odour stimulus. Horizontal grey bars represent thetime of peak amplitude of subthreshold responses. The colour code is the same as in A. C, distribution of outputresponse types: subthreshold (sub) 58%, action potentials (AP) 22% and spikelets 14%. No response (non) wasobserved in 6% of the presentations (top). Below, distribution of response types for each specific odour; colourcode as in A.



injection or odour stimulation (500 ms, 10–50 pA orsniff-like odour presentation). Notably, the frequencyof persistent firing was independent of the amplitudeof current injected to the cell or of the firing rate

evoked by the current injection (r = 0.139; Fig. 2E, topright) (Pressler & Strowbridge, 2006). In slice studiesit has been shown that persistently firing interneuronsin the GC layer mediate monosynaptic feed-forward

Figure 2. Rare odour-evoked phasic inhibition in GCsA, schematic drawing of the local GC network. The mitral cell (MC) connects to the GC via excitatory (Ex)dendro-dendritic synapses on the MC lateral dendrites and via collaterals to local interneurons (dSAC, green; In).The arrow represents olfactory drive from the olfactory nerve (ON). B, odour-evoked excitatory (grey) and inhibitory(green) synaptic currents from six different cells recorded during voltage-clamping at –75 mV (a) and +20 mV (b).Below, corresponding peri-stimulus histograms (bin = 20 ms, five trials). Red bars denote the time of odourpresentation. C, distribution of evoked and non-evoked excitatory and inhibitory odour responses, respectively. D,bar chart that shows the percentage of cells with non-evoked, excitatory-only and evoked inhibition–excitationresponses below. Example of EPSC (grey) and IPSC (green) in response to odour presentation (red bar). To illustratethe time lag of inhibition, the EPSC has been horizontally mirrored (light grey). E, in vivo recording from dSACs inwhich odour stimulation induced long-lasting spike trains at frequencies of 2–4 Hz after the odour presentation(top left). No relationship was found between the spiking frequency during current injections and the persistentspiking of these suspected dSACs (r = 0.139; top right). Bottom left, recording and raster of IPSCs after aninhibitory evoked response in a GC. Bottom right, normalized frequency of spontaneous IPSCs before and afterodour presentation in GCs that responded with evoked inhibition. The rate of IPSCs after the evoked responsewas analysed during a 1- to 5-s interval after the odour presentation.



inhibition onto GCs (Pressler & Strowbridge, 2006).These findings led us to analyse the post-stimulusfrequency of IPSCs in GCs responding with IPSC bursts.We found that the few GCs with inhibitory evokedpotentials exhibited an increase in single IPSCs frequencyafter an odour-evoked response (IPSCspont = 2.2 ± 2.9vs. IPSCafter-stim = 4.4 ± 8.4 Hz, P < 0.05; n = 32 pre-sentations in 11 cells; Fig. 2E, bottom right). This increasein IPSCs was confined to GCs with evoked inhibitoryresponses and not observed in the large group of GCs thatlacked inhibitory evoked bursts upon odour presentation(IPSCspont = 1.8 ± 1.5 vs. IPSCafter-stim = 1.28 ± 0.72 Hz,P > 0.05; n = 23 presentations in 15 cells; data not shown).

These results may suggest that dSACs with a persistentpattern synapse onto GCs.

Tonic inhibition in GCs

The GABAergic origin of IPSC events in GCs wasverified by applying gabazine (Fig. 3A). Gabazineexpectedly removed transient synaptic currents butsurprisingly also significantly decreased the holdingcurrent (�I = 24.3 ± 10.3 pA, P < 0.05, n = 4; Fig. 3A).Additionally, the current shift was associated witha reduction of the baseline peak-to-peak noise level(Fig. 3A, right panel, Gaussian widths), consistent withthe blockade of background inhibitory tonic conductance(Bright & Brickley, 2008).

Previous studies have shown that inhibitory tonicconductance is mediated by extrasynaptic GABAA

receptors, often expressing α4 and δ subunits (Mody,2001). Evidence at the mRNA level suggests that olfactorybulb GCs express extrasynaptic α4 and δ GABAA sub-units. Furthermore, GABAA α4 and δ subunit mRNAwas exclusively found in the GC layer and only on GCs(Laurie et al. 1992). To investigate the possible impact ofthese extrasynaptic GABAA receptors on GCs, we appliedthe GABAA α4/α6 subtype-selective antagonist furosemide(Korpi et al. 1995, Korpi & Luddens, 1997, Mody, 2001).Furosemide significantly reduced the baseline holdingcurrent (�I = 42.0 ± 19.3 pA; P < 0.05, n = 4; Fig. 3B),whereas no change in the waveform characteristicsand frequency was observed for synaptic inhibitoryevents (IPSCrise ctrl = 1.84 ± 0.66 vs. IPSCrise furo = 2.67± 0.81 ms, P > 0.05; IPSCdecay ctrl = 22.27 ± 3.17 vs.IPSCdecay furo = 22.75 ± 4.09 ms, P > 0.5; IPSCamp ctrl =49.25 ± 6.4 vs. IPSCamp furo = 42 ± 5.89 pA, P > 0.05,IPSCfreq . ctrl = 3.13 ± 3.18 vs. IPSCfreq . furo = 4.0 ± 4.4 Hz,P > 0.05; n = 4, Fig. 3C, overlay). Moreover, a clearincrease in the input resistance of GCs wasobserved, supporting our hypothesis that the changein holding current resulted from blockade of tonicinhibition mediated by extrasynaptic GABAA receptors

(Rctrl = 339 ± 49 vs. Rfuro 496 ± 189 m�, P < 0.05; n = 7,data not shown).

The presence of tonic inhibition in GCs in the olfactorybulb is novel and the cellular mechanism behind it isas yet unknown. In other systems the most widespreadexplanation for the origin of extrasynaptic GABAergicactivity is spillover from neighbouring synapses (Farrant& Nusser, 2005; Duguid et al. 2012). However, tonicinhibition of GCs in the cerebellum has also beenshown to involve glial cells (Lee et al. 2010). Anotherphysiologically viable option is that reversed activity ofsodium chloride symporters dynamically regulates thelevel of extrasynaptic GABA (Richerson & Wu, 2003).In accordance with previous studies, we analysed themagnitude of tonic conductance compared with thefrequency of an individual IPSC to investigate the potentialrole of spillover as a source of extrasynaptic GABA(Bright & Brickley, 2008). As no correlation was found(r = 0.03; Fig. 3C), this suggests that tonic inhibition isnot a consequence of synaptic spillover from the GCsown GABAergic input. However, it does not rule out thepossibility of spillover from the surrounding populationof GCs.

Tonic inhibition, not synaptic inhibition, affects GCactivity

To examine the impact of tonic conductance comparedwith phasic inhibitory input on GC activity an analysisof charge contribution was performed. We found that theaverage current associated with tonic conductance was3-fold greater than the average current of evoked phasicinhibitory events (32 ± 16 vs. 10 ± 9 pC s−1; n = 8 and26, respectively; Fig. 3D). Blockage of tonic inhibitionaltered the evoked output properties of GCs such thatcells with a subthreshold response to odour presentationprior to furosemide application often responded withfull-amplitude APs after applying furosemide (Fig. 4A).The proportion of responses that included full-amplitudeAPs increased from 10.2 ± 31.6 to 40.1 ± 51.6% in the pre-sence of furosemide (Fig. 4B, left; P < 0.05 paired t test;n = 10 cells, one cell per mouse, average and standarddeviations across mice; four odours presented five timeseach). Moreover, the number of APs evoked during asingle stimulated respiratory cycle increased (0.8 ± 1.2 vs.2.4 ± 3.4, P < 0.05; n = 10, Fig. 4B, right). This indicatesthat tonic conductance plays a central role in determiningthe spiking probability of GCs.

Discussion

It is striking that although GCs rarely spike, they play akey role for odour processing in the bulb (Abraham et al.2010). In the present study, we investigated synaptic inputand firing responses of GCs. We found that excitatory



synaptic currents were broadly evoked in 71% of odourpresentations. In contrast, inhibitory evoked responseswere rare and only observed in 18% of the presentations.The low occurrence of evoked inhibitory responses showsthat phasic synaptic inhibition plays only a minor role inshaping the output of GCs during odour processing. Wefind that subthreshold responses are the most commonresponse type, accounting for 58% of responses, followedby spikes (22%) and spikelets (14%).

Furthermore, we identified a tonic inhibitoryconductance in GCs and provided evidence of theinvolvement of furosemide-sensitive GABAA receptors,probably a high-affinity combination of α4 and δ sub-units (Laurie et al. 1992, Olsen & Sieghart, 2009). Thistonic inhibition showed a strong suppressive effect on theexcitability of GCs and ultimately determined whethera GC elicited a sub- or suprathreshold response duringodour-evoked sniffing. We propose that tonic inhibition

provides the GCs with the ability to shift between local(dendro-dendritic) and global release forms (Fig. 4C).

The local inhibitory network of the GC

In the present study we revealed a marked discrepancyin the occurrence of synaptically evoked excitation andinhibition in GCs. Concurrent excitation and inhibitionare observed in many neurons during network activity,but GCs display prominent excitation and a strikingpaucity of synaptic inhibition during odour presentation.Interestingly, we only observed evoked inhibition incombination with evoked excitation. Moreover, ourrecordings of GCs revealed that evoked synaptic inhibitionwas followed by an increase in the rate of spontaneousIPSCs >1000 ms after odour presentation. Assumingthat dSACs are the source of phasic inhibition, the

Figure 3. Tonic inhibition of GCs is mediated by furosemide-sensitive GABAA receptors and independentof phasic synaptic GABAergic transmissionA, representative traces (left), time course of the experiment (middle) and all-points histograms from before andafter blocking the GABAergic currents with gabazine. Note the shift in the holding current (�I = 24.3 ± 10.3 pA,P < 0.05; n = 4) and reduction in the current variance. B, as in A with furosemide (purple; �I = 42.0 ± 19.3 pA,P < 0.05; n = 4). C, left, overlay of control and furosemide sIPSCs (average of 10 events). Right, tonic conductancevs. rate of sIPSCs from each individual cell (r = 0.03). Data points are from blockade by both gabazine andfurosemide. D, comparison of charge transfer from evoked synaptic and tonic currents measured over 1000 ms(evoked synaptic = 0.01 ± 0.009 pC s−1 (n = 26) vs. tonic = 0.032 ± 0.016 pC s−1 (n = 10), P < 0.01).



fact that a GC with phasic inhibition alone was neverencountered suggests that the dSACs receive input fromthe same population of principal neurons as the GCs.As a consequence, the same AP that back-propagatesinto the lateral mitral cell dendrite and excites the GCmay also evoke phasic inhibition via activation of aninhibitory dSAC that synapses onto the GC. Thus, viainhibition of the GC the principal neuron activates itsown disinhibition. Among the interneurons in the GC

layer, the Blanes cells, a subtype of dSACs, mediatefeed-forward synaptic inhibition onto GCs and respondin vitro with long trains of AP in response to briefdepolarization. A more hyperpolarized threshold levelof AP initiation compared with GCs also characterizesBlanes cells, characteristics observed in the dSACs werecorded as well. Interestingly, we also found that theprobability of obtaining an evoked inhibitory response(18%) was coherent with the likelihood of obtaining

Figure 4. Blockade of tonic GABAergic current increases GCexcitabilityA, odour-evoked responses in control (grey) and after application offurosemide (blue). Below, rasters from five odour trials. B, left,distribution of output response types in control and with furosemide:subthreshold (grey), spikelets (black) and action potential (blue).Right, average number of spikes in five trials during an odour-evokedsniff in control and with furosemide (Ctrl vs. furosemide: 1.1 ± 2.2vs. 3.5 ± 2.8 APs, P < 0.01; n = 35). C, schematic drawingillustrating how the level of tonic inhibition could regulate the extentof inhibition mediated by the GC (black) on to the mitral cells.Principal neurons are illustrated via their long lateral dendrites (greyand blue: lateral inhibited). We propose that the ambient level oftonic inhibition can act as a regulator between recurrent and lateralinhibition mediated by the GC. A back-propagating AP in the mitralcells lateral dendrite will give rise to local recurrent inhibition onlyduring high levels of tonic inhibition (C, upper). Under low tonicinhibitory conditions a similar input from the mitral cell might causeglobal lateral inhibition (C, lower); see Discussion.



Blanes cell/GC monosynaptically coupled pairs in slices(Pressler & Strowbridge, 2006).

Lastly, inhibition of GCs may be of local origin, althoughinput from centrifugal inhibitory fibres is also possible(Kunze et al. 1992, Gracia-Llanes et al. 2010).

Tonic inhibition regulates the output properties ofGCs

The discovery of tonic inhibition in GCs may explainthe hyperpolarized resting membrane potential, the lowspike rate and why GCs need strong, synchronous inputto reach threshold (Cang & Isaacson, 2003, Margrie &Schaefer, 2003). Our findings showed that tonic inhibitionhas a strong impact on the output of GCs and dominatesover phasic inhibition. Although loop diuretic drugs suchas furosemide have been shown to block the potassiumchloride cotransporter 2 (KCC2) in rat hippocampal invitro studies at concentrations of 0.5 and 1 mM (Viitanenet al. 2010), our results do not show indications ofsuch interaction, as no change of IPSC waveforms wereobserved (Fig. 3C). The fact that odour-evoked sub-threshold responses were changed to full-amplitude APs,after application of furosemide, suggests that the dynamiclevel of tonic inhibition regulates the extent of inhibition,i.e. possibly from recurrent to lateral (Fig. 4C).

It is possible that high levels of tonic inhibitoryconductance maintain olfactory GCs in a state that favourslocal graded regenerative responses in dendritic spinesand branches to enrich and differentiate GABA release,similar to the isolated signal processing that occurs inamacrine cells (Grimes et al. 2010). The morphologyof GCs, distribution of release sites and bidirectionaldendro-dentritic synapses appear well suited to allowrelease sites in the same neuron to be regulated by thelevel of tonic inhibition, thereby playing a key role in thedynamics of dense synchronous synaptic release versusa temporal spatial GABA release from the GC dendritictree. Thus, strong shunting via high tonic conductancewould limit activation to local dendritic branches only.In contrast, low levels of tonic inhibition would permitactivation in local branches/spines to spread throughoutthe dendritic tree causing lateral (global) inhibition of theconnected population of mitral cells.

We propose that the spatiotemporal evolution of circuitactivity during odour discrimination depends on theseproperties provided by tonic extrasynaptic inhibition inGCs.

The source of GABA that activates extrasynapticreceptors is unknown. There was no clear relationshipbetween the magnitude of tonic conductance andspontaneous IPSC frequency in individual GCs,supporting the hypothesis that synaptic and extrasynapticGABAA receptors are recruited by GABA from separatesources (Bright & Brickley, 2008). Yet, this does not entirely

rule out spillover as a source of extrasynaptic GABA. Oneobvious possibility is the numerous and densely packedrelease sites from neighbouring GCs, which could providea source of volume-conducted GABA in the extracellularspace.

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Author contributions

C.L. performed all experiments. K.A. and C.L performed the dataanalysis with help from M.L. M.L. provided laboratory facilitiesand inputs to the second round of experiments, data analysisand figures. C.L. and K.A. wrote the manuscript, with inputsfrom M.L. All authors approve the final version for publication.

Acknowledgements

We thank Elad Ganmor, Jørn Hounsgaard and Andreas T.Schaefer for commenting on the manuscript. This work wassupported by The Danish Council for Independent Research(K.A.), The Carlsberg Foundation, The Oticon Foundation andThe Lundbeck Foundation.


Tonic inhibition sets the state of excitability in olfactory bulb granule cells

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