Formation and function of neural circuitry in the olfactory bulb of mice with reduced afferent spontaneous activity

Abstract

Nei sistemi sensoriali, i neuroni periferici proiettano i loro assoni in specifici loci del cervello. La segregazione spaziale delle afferenze sensoriali provvede a creare mappe topografiche che definiscono la qualit\ue0 e la localizzazione di complessi stimoli sensoriali. L\u2019attivit\ue0 elettrica gioca un ruolo chiave nella formazione di specifici contatti sinaptici tra i neuroni, sebbene resti ancora da definire il tipo di attivit\ue0 richiesta. In particolare il ruolo dell\u2019attivit\ue0 elettrica spontanea nell\u2019organizzazione topografica del sistema olfattivo, non \ue8 noto. Per rispondere a questa domanda abbiamo studiato il ruolo dell\u2019attivit\ue0 elettrica spontanea nella formazione e nella funzione dei circuiti neurali nel bulbo olfattivo. Per raggiungere questo obiettivo abbiamo utilizzato una linea di topi geneticamente modificati, nei quali l\u2019attivit\ue0 afferente spontanea \ue8 ridotta a causa della sovra-espressione di un canale potassio inward rectifier (Kir2.1), in tutti i neuroni olfattivi sensoriali (i topi Kir2.1). Abbiamo analizzato la formazione della mappa sensoriale, in particolare se la convergenza dei neuroni sensoriali esprimenti il medesimo recettore olfattivo avveniva correttamente nei topi Kir2.1 La convergenza dei neuroni sensoriali in specifici loci del bulbo olfattivo, che porta alla formazione di glomeruli omogenei, cio\ue9 glomeruli formati esclusivamente da assoni esprimenti lo stesso recettore olfattivo, \ue8 una caratteristica critica della mappa sensoriale. Infatti i glomeruli definiscono le unit\ue0 funzionali o colonne odorose del sistema. Abbiamo trovato che in assenza di attivit\ue0 spontanea, gli assoni dei neuroni sensoriali non convergono a formare un unico glomerulo ma proiettano in molti siti dando luogo a ulteriori glomeruli. Questi addizionali glomeruli sono caratterizzati da una organizzazione eterogenea, risultano cio\ue8 formati da assoni di neuroni sensoriali esprimenti recettori olfattivi diversi. Per capire se l\u2019attivit\ue0 afferente spontanea potesse avere un ruolo anche sulle cellule postsinaptiche del bulbo olfattivo, abbiamo analizzato le cellule mitrali, i principali neuroni di output, e le cellule dei granuli, i principali neuroni inibitori, del bulbo olfattivo. Analizzando lo sviluppo morfologico del dendrite apicale delle cellule mitrali non abbiamo trovato alcuna differenza significativa nei topi Kir2.1 rispetto ai controlli. Per quanto concerne le cellule dei granuli, studiando la neurogenesi e la migrazione delle cellule dei granuli di nuova generazione, non abbiamo riscontrato differenze significative nei topi Kir2.1 rispetto ai controlli. Tuttavia l\u2019analisi morfologica dell\u2019arborizzazione dendritica delle cellule dei granuli ha messo in evidenza una ridotta densit\ue0 di filopodi/spine nei topi Kir2.1 rispetto ai controlli. Per analizzare le conseguenze funzionali di queste alterazioni anatomiche abbiamo eseguito specifici test comportamentali. I dati che abbiamo ottenuto indicano chiaramente che i topi Kir2.1 non erano in grado di discriminare tra due odori che attivano glomeruli che hanno distribuzione spaziale molto simile, quali gli enantiomeri. Tuttavia i topi Kir2.1 mantenevano la capacit\ue0 di distinguere odori che attivano glomeruli posti in aree molto diverse del bulbo, quali l\u2019acido 2-metilbutirrico e l\u2019acido ciclobutancarbossilico (2Mb e CB). Dato l\u2019elevato grado di plasticit\ue0 del sistema olfattivo, ci siamo chiesti se la manipolazione dell\u2019attivit\ue0 elettrica in et\ue0 adulta poteva influenzare la mappa sensoriale. Sfruttando la possibilit\ue0 di indurre l\u2019espressione del gene Kir2.1 in momenti diversi della vita dell\u2019animale, abbiamo fatto esprimere il gene Kir2.1 solo in animali adulti per 4 settimane. Abbiamo trovato che l\u2019espressione del gene Kir2.1 in animali adulti alterava l\u2019organizzazione della mappa sensoriale, cio\ue9 la specifica convergenza degli assoni dei neuroni sensoriali nel bulbo olfattivo. I dati ottenuti indicano che l\u2019assenza di attivit\ue0 spontanea nell\u2019et\ue0 adulta causa una \u201cregressione\u201c nell\u2019organizzazione dei glomeruli. Abbiamo infatti trovato un elevato numero di glomeruli eterogenei che coesistevano coi principali glomeruli omogenei. I nostri dati suggeriscono che l\u2019attivit\ue0 elettrica spontanea \ue8 richiesta per lo sviluppo e il mantenimento della mappa sensoriale. Inoltre abbiamo trovato che le alterazioni morfologiche della circuiteria neuronale nel bulbo olfattivo contribuiscono ad alterare il comportamento olfattivo.In the sensory systems, peripheral neurons project axons in specific loci of the brain. The spatial segregation of the sensory afferents provides topographic maps that define the quality and the location of complex sensory stimuli. Electrical activity plays a critical role in the formation of specific synaptic contacts among neurons, although the type of activity required remains a matter of significant debate. In particular the role of spontaneous activity in the formation of the topographic organization of the olfactory system remains unknown. To address this question we investigated the role of spontaneous electrical activity in circuit formation and function in the olfactory bulb. To accomplish this goal, we took advantage of a line of mice engineered to have very little afferent spontaneous activity due to the over expression of the inward rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). We analyzed the formation of the sensory map, in particular whether the convergence of olfactory sensory neurons expressing the same odorant receptor took place properly in mice with reduced afferent spontaneous activity. The conflation of sensory axons to form homogeneous glomeruli, i.e. glomeruli formed exclusively by axons expressing the same olfactory receptor, in specific loci of the olfactory bulb is a critical feature of the sensory map that in turn defines functional units, i.e. odor columns. We found that in absence of spontaneous activity, the convergence of sensory neurons to form homogenous glomeruli took place but it was coarser than in controls. In particular we observed axons mistargeting that resulted in multiple heterogeneous glomeruli that persist also in adulthood. To ascertain the role of spontaneous activity on the post synaptic elements of the olfactory bulb, we analyzed the mitral cells, the principal output neurons, and the granule cells, the major component of the inhibitor interneurons of the olfactory bulb. We found no difference in the developmental refinement of the apical dendrite of mitral cells in Kir2.1 and in control mice. The neurogenesis and the migration of granule cells was unaltered. However the filopodia-spine density on the dendritic tree of the granule cells was significantly reduced in Kir2.1 mice. To analyze the functional outcome of these anatomical alterations, we performed behaviour experiments. We demonstrated that Kir2.1 mice were unable to discriminate between two odors, such as couple of enantiomers, that elicit very similar spatial patterns of activated glomeruli, while retained the ability to differentiate between odorants, such as 2-methylbutyric acid and cyclobutanecarboxylic acid (2Mb and CB) that activate patterns of glomeruli spatially very distinct. Due to the high degree of plasticity of the olfactory system, we asked whether manipulation of electrical activity in adulthood could affect the already refined neural circuitry in the olfactory bulb. Taking advantage of the inducible nature of the Kir2.1 construct, we allowed the expression of the Kir2.1 channels only in adulthood, for 4 weeks. We found that the expression of the Kir2.1 channel in adults disrupted the organization of the sensory map, namely the convergence of olfactory sensory neuron axons. The absence of spontaneous afferent activity in adults induced a regression in the glomeruli organization. We found supernumerary heterogeneous glomeruli that coexist with the main homogeneous glomeruli. In the sensory systems, peripheral neurons project axons in specific loci of the brain. The spatial segregation of the sensory afferents provides topographic maps that define the quality and the location of complex sensory stimuli. Electrical activity plays a critical role in the formation of specific synaptic contacts among neurons, although the type of activity required remains a matter of significant debate. In particular the role of spontaneous activity in the formation of the topographic organization of the olfactory system remains unknown. To address this question we investigated the role of spontaneous electrical activity in circuit formation and function in the olfactory bulb. To accomplish this goal, we took advantage of a line of mice engineered to have very little afferent spontaneous activity due to the over expression of the inward rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). We analyzed the formation of the sensory map, in particular whether the convergence of olfactory sensory neurons expressing the same odorant receptor took place properly in mice with reduced afferent spontaneous activity. The conflation of sensory axons to form homogeneous glomeruli, i.e. glomeruli formed exclusively by axons expressing the same olfactory receptor, in specific loci of the olfactory bulb is a critical feature of the sensory map that in turn defines functional units, i.e. odor columns. We found that in absence of spontaneous activity, the convergence of sensory neurons to form homogenous glomeruli took place but it was coarser than in controls. In particular we observed axons mistargeting that resulted in multiple heterogeneous glomeruli that persist also in adulthood. To ascertain the role of spontaneous activity on the post synaptic elements of the olfactory bulb, we analyzed the mitral cells, the principal output neurons, and the granule cells, the major component of the inhibitor interneurons of the olfactory bulb. We found no difference in the developmental refinement of the apical dendrite of mitral cells in Kir2.1 and in control mice. The neurogenesis and the migration of granule cells was unaltered. However the filopodia-spine density on the dendritic tree of the granule cells was significantly reduced in Kir2.1 mice. To analyze the functional outcome of these anatomical alterations, we performed behaviour experiments. We demonstrated that Kir2.1 mice were unable to discriminate between two odors, such as couple of enantiomers, that elicit very similar spatial patterns of activated glomeruli, while retained the ability to differentiate between odorants, such as 2-methylbutyric acid and cyclobutanecarboxylic acid (2Mb and CB) that activate patterns of glomeruli spatially very distinct. Due to the high degree of plasticity of the olfactory system, we asked whether manipulation of electrical activity in adulthood could affect the already refined neural circuitry in the olfactory bulb. Taking advantage of the inducible nature of the Kir2.1 construct, we allowed the expression of the Kir2.1 channels only in adulthood, for 4 weeks. We found that the expression of the Kir2.1 channel in adults disrupted the organization of the sensory map, namely the convergence of olfactory sensory neuron axons. The absence of spontaneous afferent activity in adults induced a regression in the glomeruli organization. We found supernumerary heterogeneous glomeruli that coexist with the main homogeneous glomeruli. All together our data suggest that spontaneous activity is required for the developmental refinement and maintenance of the sensory map. Furthermore we found that the unrefined connectivity of the neural circuitry of the olfactory bulb could affect olfactory discrimination behaviour