Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Université de Bourgogne

The septal organ, a distinct chemosensory organ observed in the mammalian nose, is essentially a small island of olfactory neuroepithelium located bilaterally at the ventral base of the nasal septum. Virtually nothing is known about its physiological properties and function. To understand the nature of the sensory neurons in this area, we studied the mechanisms underlying olfactory signal transduction in these neurons. The majority of the sensory neurons in the septal organ express olfactory-specific G-protein and adenylyl cyclase type III, suggesting that the cAMP signaling pathway plays a critical role in the septal organ as in the main olfactory epithelium (MOE). This is further supported by patch-clamp recordings from individual dendritic knobs of the sensory neurons in the septal organ. Odorant responses can be mimicked by an adenylyl cyclase activator and a phosphodiesterase inhibitor, and these responses can be blocked by an adenylyl cyclase inhibitor. There is a small subset of cells in the septal organ expressing a cGMP-stimulated phosphodiesterase (phosphodiesterase 2), a marker for the guanylyl cyclase-D subtype sensory neurons identified in the MOE. The results indicate that the septal organ resembles the MOE in major olfactory signal transduction pathways, odorant response properties, and projection to the main olfactory bulb. Molecular and functional analysis of the septal organ, which constitutes approximately 1% of the olfactory epithelium, will provide new insights into the organization of the mammalian olfactory system and the unique function this enigmatic organ may serve.

Olfactory signal transduction in the mouse septal organ.

A glomerulus in the mammalian olfactory bulb receives axonal inputs from olfactory sensory neurons (OSNs) that express the same odorant receptor (OR). Glomeruli are generally thought to represent functional units of olfactory coding, but there are no data on the electrophysiological properties of OSNs that express the same endogenous OR. Here, using patch clamp recordings in an intact epithelial preparation, we directly measured the transduction currents and receptor potentials from the dendritic knobs of mouse OSNs that express the odorant receptor MOR23 along with the green fluorescent protein. All of the 53 cells examined responded to lyral, a known ligand for MOR23. There were profound differences in response kinetics, particularly in the deactivation phase. The cells were very sensitive to lyral, with some cells responding to as little as 10 nM. The dynamic range was unexpectedly broad, with threshold and saturation in individual cells often covering three log units of lyral concentration. The potential causes and biological significance of this cellular heterogeneity are discussed. Patch clamp recording from OSNs that express a defined OR provides a powerful approach to investigate the sensory inputs to individual glomeruli.

Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.

Most sensory systems are primarily specialized to detect one sensory modality. Here we report that olfactory sensory neurons (OSNs) in the mammalian nose can detect two distinct modalities transmitted by chemical and mechanical stimuli. As revealed by patch-clamp recordings, many OSNs respond not only to odorants, but also to mechanical stimuli delivered by pressure ejections of odor-free Ringer solution. The mechanical responses correlate directly with the pressure intensity and show several properties similar to those induced by odorants, including onset latency, reversal potential and adaptation to repeated stimulation. Blocking adenylyl cyclase or knocking out the cyclic nucleotide-gated channel CNGA2 eliminates the odorant and the mechanical responses, suggesting that both are mediated by a shared cAMP cascade. We further show that this mechanosensitivity enhances the firing frequency of individual neurons when they are weakly stimulated by odorants and most likely drives the rhythmic activity (theta oscillation) in the olfactory bulb to synchronize with respiration.

Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.

Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth Mamestra brassicae.

In mammals, the sense of smell is modulated by the status of satiety, which is mainly signaled by blood-circulating peptide hormones. However, the underlying mechanisms linking olfaction and food intake are poorly understood. Here we investigated the effects of two anorectic peptides, insulin and leptin, on the functional properties of olfactory sensory neurons (OSNs). Using patch-clamp recordings, we analyzed the spontaneous activity of rat OSNs in an in vitro intact epithelium preparation. Bath perfusion of insulin and leptin significantly increased the spontaneous firing frequency in 91.7% (n = 24) and 75.0% (n = 24) of the cells, respectively. When the activity was electrically evoked, both peptides shortened the latency to the first action potential by approximately 25% and decreased the interspike intervals by approximately 13%. While insulin and leptin enhanced the electrical excitability of OSNs in the absence of odorants, they surprisingly reduced the odorant-induced activity in the olfactory epithelium. Insulin and leptin decreased the peak amplitudes of isoamyl acetate-induced electroolfactogram (EOG) signals to 46 and 38%, respectively. When measured in individual cells by patch-clamp recordings, insulin and leptin decreased odorant-induced transduction currents and receptor potentials. Therefore by increasing the spontaneous activity but reducing the odorant-induced activity of OSNs, an elevated insulin and leptin level (such as after a meal) may result in a decreased global signal-to-noise ratio in the olfactory epithelium, which matches the smell ability to the satiety status.

Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin.

The sense of smell deteriorates in normal aging, but the underling mechanisms are still elusive. Here we investigated age-related alterations in expression patterns of odorant receptor (OR) genes and functional properties of olfactory sensory neurons (OSNs)-2 critical factors that define the odor detection threshold in the olfactory epithelium. Using in situ hybridization for 9 representative OR genes, we compared the cell densities of each OR in coronal nose sections at different ages (3-27 months). The cell density for different ORs peaked at different time points and a decline was observed for 6 of 9 ORs at advanced ages. Using patch clamp recordings, we then examined the odorant responses of individual OSNs coexpressing a defined OR (MOR23) and green fluorescent protein. The MOR23 neurons recorded from aged animals maintained a similar sensitivity and dynamic range in response to the cognate odorant (lyral) as those from younger mice. The results indicate that although the cell densities of OSNs expressing certain types of ORs decline at advanced ages, individual OSNs can retain their sensitivity. The implications of these findings in age-related olfactory deterioration are discussed.

Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.

L&#39;attuale modello di consenso nell&#39;olfatto dei mammiferi &egrave; che il rilevamento di milioni di odorizzazione richiede un gran numero di recettori odorant (ORs) e che ogni OR interagisce selettivamente con un piccolo sottoinsieme di odorizzazione, che in genere sono correlati in struttura. Riportiamo le propriet&agrave; di risposta odorant di un OR che devia da questo modello: SR1, un mouse o che &egrave; abbondantemente espresso nei neuroni sensoriali dell&#39;organo del setto e anche dell&#39;epitelio olfattivo principale. Registrazioni di patch-clamp rivelano che neuroni sensoriali olfattivi (OSNs) che esprimono SR1 rispondano a molti, odorizzazione strutturalmente indipendenti e sopra una gamma ampia di concentrazione. La maggior parte delle OSNs che esprimono un locus gene targeting SR1 che manca la sequenza codifica SR1 non mostrano questa ampia capacit&agrave; di risposta. Trasferimento del gene nel sistema di espressione eterologa Hana3A conferma il profilo di ampia risposta di SR1. Ci possono essere altri mouse ORs con tali profili ampia risposta.

SR1, un recettore odorant mouse con un profilo insolitamente ampia risposta.

&#x5F53;&#x524D;&#x5171;&#x8BC6;&#x6A21;&#x578B;&#x5728;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x55C5;&#x89C9;&#x662F;&#x6C14;&#x5473;&#x7684;&#x6C14;&#x5473;&#x7684;&#x6570;&#x4EE5;&#x767E;&#x4E07;&#x8BA1;&#x68C0;&#x6D4B;&#x9700;&#x8981;&#x5927;&#x91CF;&#x7684;&#x6C14;&#x5473;&#x53D7;&#x4F53; (ORs) &#x548C;&#x6BCF;&#x4E2A;&#x6216;&#x6709;&#x9009;&#x62E9;&#x6027;&#x5730;&#x4EA4;&#x4E92;&#xFF0C;&#x901A;&#x5E38;&#x5728;&#x7ED3;&#x6784;&#x4E0A;&#x76F8;&#x5173;&#x7684;&#x4E00;&#x5C0F;&#x90E8;&#x5206;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x6216;&#x6C14;&#x5473;&#x54CD;&#x5E94;&#x7279;&#x6027;&#xFF0C;&#x504F;&#x79BB;&#x6B64;&#x6A21;&#x578B;&#xFF1A; SR1&#xFF0C;&#x9F20;&#x6807;&#x6216;&#x8005;&#xFF0C;&#x6E05;&#x695A;&#x5730;&#x8868;&#x793A;&#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x95F4;&#x9694;&#x7684;&#x673A;&#x5173;&#x548C;&#x4E3B;&#x8981;&#x55C5;&#x4E0A;&#x76AE;&#x3002;&#x819C;&#x7247;&#x94B3;&#x5F55;&#x97F3;&#x63ED;&#x793A;&#x55C5;&#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143; &#xFF08;OSNs&#xFF09; &#x8868;&#x793A; SR1 &#x56DE;&#x5E94;&#x8BB8;&#x591A;&#xFF0C;&#x7ED3;&#x6784;&#x4E0A;&#x4E0D;&#x76F8;&#x5173;&#x7684;&#x6C14;&#x5473;&#xFF0C;&#x548C;&#x5BBD;&#x6D53;&#x5EA6;&#x8303;&#x56F4;&#x5185;&#x3002;&#x5927;&#x591A;&#x6570; OSNs &#x8868;&#x793A;&#x7F3A;&#x4E4F; SR1 &#x7F16;&#x7801;&#x5E8F;&#x5217;&#x7684;&#x57FA;&#x56E0;&#x9776;&#x5411; SR1 &#x8F68;&#x8FF9;&#x4E0D;&#x4F1A;&#x663E;&#x793A;&#x6B64;&#x5E7F;&#x6CDB;&#x7684;&#x54CD;&#x5E94;&#x80FD;&#x529B;&#x3002;&#x5F02;&#x6E90;&#x8868;&#x8FBE;&#x7CFB;&#x7EDF; Hana3A &#x4E2D;&#x7684;&#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#x786E;&#x8BA4;&#x7684;&#x5E7F;&#x6CDB;&#x54CD;&#x5E94; SR1 &#x7684;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x3002;&#x53EF;&#x80FD;&#x6709;&#x5176;&#x4ED6;&#x9F20;&#x6807;&#x53E3;&#x670D;&#x8865;&#x6DB2;&#x76D0;&#x4E0E;&#x8FD9;&#x79CD;&#x5E7F;&#x6CDB;&#x54CD;&#x5E94;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x3002;

SR1&#xFF0C;&#x9F20;&#x6807;&#x6C14;&#x5473;&#x53D7;&#x4F53;&#x5177;&#x6709;&#x5F02;&#x4E4E;&#x5BFB;&#x5E38;&#x7684;&#x5E7F;&#x6CDB;&#x54CD;&#x5E94;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x3002;

The current consensus model in mammalian olfaction is that the detection of millions of odorants requires a large number of odorant receptors (ORs) and that each OR interacts selectively with a small subset of odorants, which are typically related in structure. Here, we report the odorant response properties of an OR that deviates from this model: SR1, a mouse OR that is abundantly expressed in sensory neurons of the septal organ and also of the main olfactory epithelium. Patch-clamp recordings reveal that olfactory sensory neurons (OSNs) that express SR1 respond to many, structurally unrelated odorants, and over a wide concentration range. Most OSNs expressing a gene-targeted SR1 locus that lacks the SR1 coding sequence do not show this broad responsiveness. Gene transfer in the heterologous expression system Hana3A confirms the broad response profile of SR1. There may be other mouse ORs with such broad response profiles.

SR1, a mouse odorant receptor with an unusually broad response profile.

Le mod&#xE8;le du consensus actuel dans l&#x26;#39;olfaction chez les mammif&#xE8;res, c&#x26;#39;est que la d&#xE9;tection de millions de mol&#xE9;cules odorantes n&#xE9;cessite un grand nombre de r&#xE9;cepteurs olfactifs (OR) et que chaque OU interagit s&#xE9;lectivement avec un petit sous-ensemble de substances odorantes, qui sont g&#xE9;n&#xE9;ralement li&#xE9;s &#xE0; la structure. Ici, nous pr&#xE9;sentons les propri&#xE9;t&#xE9;s odorantes de r&#xE9;ponse de une OU qui s&#x26;#39;&#xE9;carte de ce mod&#xE8;le: SR1, une souris ou qui est abondamment exprim&#xE9;e dans les neurones sensoriels de l&#x26;#39;organe septal et aussi de l&#x26;#39;&#xE9;pith&#xE9;lium olfactif principal. Patch-clamp r&#xE9;v&#xE8;lent que les neurones olfactifs sensoriels (ARS) qui expriment SR1 face &#xE0; beaucoup, odorants structurellement ind&#xE9;pendants, et sur une large gamme de concentration. La plupart des ARS exprimant un locus du g&#xE8;ne cibl&#xE9; SR1 qui manque de la s&#xE9;quence de codage SR1 ne pr&#xE9;sentent pas cette r&#xE9;activit&#xE9; large. Le transfert de g&#xE8;nes dans le syst&#xE8;me d&#x26;#39;expression h&#xE9;t&#xE9;rologue Hana3A confirme le profil de r&#xE9;ponse &#xE9;ventail de SR1. Il peut y avoir d&#x26;#39;autres RUP souris avec ces profils de r&#xE9;ponse larges.

SR1, un r&#xE9;cepteur olfactif de souris avec un profil de r&#xE9;ponse exceptionnellement large

Das aktuelle Modell der Konsens in S&auml;ugetieren Olofaktion ist, dass die Erkennung von Millionen von Ger&uuml;chen eine gro&szlig;e Anzahl von Odorant-Rezeptoren (ORs) erfordert und dass jedes OR interagiert selektiv mit einer kleinen Teilmenge von Ger&uuml;chen, die in der Regel in Struktur verbunden sind. Hier berichten wir die Odoriermittel Antwort Eigenschaften des OR, die weicht von diesem Modell: SR1, eine Maus oder, das ist reichlich, ausgedr&uuml;ckt in sensorischen Neuronen Septumdefekt Orgel und auch die wichtigsten olfaktorischen Epithel. Patch-Clamp-Aufnahmen zeigen, dass zu viele, nicht strukturell verwandten Geruchsstoffe und &uuml;ber einen weiten Konzentrationsbereich olfaktorische sensorische Neuronen (OSNs), die SR1 Ausdr&uuml;cken zu reagieren. Die meisten OSNs Ausdruck einer gen-bezogene SR1-Locus, die die SR1 kodierende Sequenz fehlt Anzeigen dieser breiten Reaktionsf&auml;higkeit nicht. Gentransfer in die heterologen Expressionssystem Hana3A best&auml;tigt das umfassende Antwort-Profil von SR1. M&ouml;glicherweise gibt es andere Maus-ORs mit solch breiten Antwort-Profilen.

SR1, ein Maus-Odorant-Rezeptor mit ein ungew&ouml;hnlich breites Antwort-Profil.

&#x54FA;&#x4E73;&#x985E;&#x306E;&#x55C5;&#x899A;&#x306E;&#x73FE;&#x5728;&#x306E;&#x30B3;&#x30F3;&#x30BB;&#x30F3;&#x30B5;&#x30B9;&#x30E2;&#x30C7;&#x30EB;&#x306F;&#x3001;&#x5302;&#x3044;&#x7269;&#x8CEA;&#x306E;&#x6570;&#x767E;&#x4E07;&#x306E;&#x691C;&#x51FA;&#x306F;&#x3001;&#x55C5;&#x899A;&#x53D7;&#x5BB9;&#x4F53;&#xFF08;ORS&#xFF09;&#x306E;&#x591A;&#x6570;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3067;&#x3042;&#x308B;&#x5404;OR&#x901A;&#x5E38;&#x306F;&#x69CB;&#x9020;&#x4F53;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x308B;&#x5302;&#x3044;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x9078;&#x629E;&#x7684;&#x306B;&#x4F5C;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x304B;&#x3089;&#x3001;&#x3042;&#x308B;&#x3044;&#x306F;&#x305D;&#x306E;&#x305A;&#x308C;&#x306E;&#x5302;&#x3044;&#x5FDC;&#x7B54;&#x7279;&#x6027;&#x3092;&#x5831;&#x544A;&#x3059;&#x308B;&#xFF1A;SR1&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x3084;&#x8C4A;&#x5BCC;&#x9694;&#x81D3;&#x5668;&#x306E;&#x3082;&#x4E3B;&#x55C5;&#x4E0A;&#x76AE;&#x306E;&#x611F;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x767A;&#x73FE;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x30D1;&#x30C3;&#x30C1;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x8A18;&#x9332;&#x306F;&#x3001;&#x30A8;&#x30AF;&#x30B9;&#x30D7;&#x30EC;&#x30B9;SR1&#x304C;&#x591A;&#x304F;&#x3001;&#x69CB;&#x9020;&#x7684;&#x306B;&#x7121;&#x95A2;&#x4FC2;&#x306A;&#x5302;&#x3044;&#x7269;&#x8CEA;&#x306B;&#x5FDC;&#x7B54;&#x3059;&#x308B;&#x55C5;&#x7D30;&#x80DE;&#xFF08;OSNs&#xFF09;&#x306F;&#x3001;&#x5E83;&#x3044;&#x6FC3;&#x5EA6;&#x7BC4;&#x56F2;&#x306B;&#x308F;&#x305F;&#x3063;&#x3066;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002; SR1&#x30B3;&#x30FC;&#x30C9;&#x914D;&#x5217;&#x3092;&#x6B20;&#x3044;&#x3066;&#x3044;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x6A19;&#x7684;&#x3068;SR1&#x306E;&#x8ECC;&#x8DE1;&#x3092;&#x8868;&#x73FE;&#x3059;&#x308B;&#x6700;&#x3082;OSNs&#x306F;&#x3001;&#x3053;&#x306E;&#x5E83;&#x7BC4;&#x306A;&#x53CD;&#x5FDC;&#x6027;&#x3092;&#x793A;&#x3055;&#x306A;&#x3044;&#x3002;&#x7570;&#x7A2E;&#x767A;&#x73FE;&#x30B7;&#x30B9;&#x30C6;&#x30E0;Hana3A&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x306F;&#x3001;SR1&#x306E;&#x5E83;&#x7BC4;&#x306A;&#x5FDC;&#x7B54;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x3088;&#x3046;&#x306A;&#x5E83;&#x7BC4;&#x306A;&#x5FDC;&#x7B54;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3092;&#x6301;&#x3064;&#x4ED6;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x8AD6;&#x7406;&#x548C;&#x304C;&#x3042;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

SR1&#x3001;&#x7570;&#x5E38;&#x306B;&#x5E83;&#x3044;&#x5FDC;&#x7B54;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3092;&#x6301;&#x3064;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x55C5;&#x899A;&#x53D7;&#x5BB9;&#x4F53;

&#xD3EC;&#xC720;&#xB958; &#xD6C4;&#xAC01;&#xC774; &#xD604;&#xC7AC; &#xD569;&#xC758; &#xBAA8;&#xB378;&#xC740; odorants&#xC758; &#xC218;&#xBC31;&#xB9CC;&#xC758; &#xAC80;&#xCD9C; odorant &#xC218;&#xC6A9; &#xCCB4; (ORs)&#xC758; &#xB2E4; &#xC218; &#xD544;&#xC694; &#xD558; &#xACE0; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xAD6C;&#xC870;&#xC5D0; &#xAD00;&#xB828; &#xB41C; odorants&#xC758; &#xC791;&#xC740; &#xD558;&#xC704; &#xC9D1;&#xD569;&#xC744; &#xAC00;&#xC9C4; &#xAC01; &#xB610;&#xB294; &#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; &#xC791;&#xC6A9; &#xD558;. &#xBCF4;&#xACE0;&#xC11C;&#xB294; OR&#xC758; odorant &#xC751;&#xB2F5; &#xC18D;&#xC131;&#xC744; &#xC5EC;&#xAE30;&#xC5D0;, &#xC6B0;&#xB9AC;&#xB294;&#xC774; &#xBAA8;&#xB378;&#xC5D0;&#xC11C;&#xC774; &#xC0C1;&#xC790;: s r 1&#xC744; &#xB9C8;&#xC6B0;&#xC2A4;&#xB97C; &#xB2E4;&#xB7C9; septal &#xC624;&#xB974;&#xAC04;&#xC758; &#xACE0;&#xB3C4; &#xC8FC; &#xD6C4; &#xAC01; &#xC0C1;&#xD53C;&#xC758; &#xAC10;&#xAC01; &#xC2E0;&#xACBD; &#xC138;&#xD3EC; &#xB2E8;&#xC704;&#xB85C; &#xB610;&#xB294;. &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504; &#xB179;&#xC74C; &#xACF5;&#xAC1C; &#xD6C4; &#xAC01; &#xAC10;&#xAC01; &#xB274;&#xB7F0; (OSNs) s r 1&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xB9CE;&#xC740;, &#xAD6C;&#xC870;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xB828; &#xC5C6;&#xB294; odorants &#xD558; &#xACE0; &#xB113;&#xC740; &#xB18D;&#xB3C4; &#xBC94;&#xC704; &#xB300;&#xC751;. SR1 &#xCF54;&#xB529; &#xC2DC;&#xD000;&#xC2A4; &#xBD80;&#xC871; &#xC720;&#xC804;&#xC790; &#xD0C0;&#xAC9F; SR1 &#xADA4;&#xC801;&#xC744; &#xD45C;&#xD604; &#xD558;&#xB294; &#xB300;&#xBD80;&#xBD84;&#xC758; Osns&#xB294;&#xC774; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC751;&#xB2F5; &#xC18D;&#xB3C4; &#xD45C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xC774;&#xB3D9; heterologous &#xC2DD; &#xC2DC;&#xC2A4;&#xD15C; hana3a&#xC5D0;&#xC5D0;&#xC11C; s r 1&#xC758; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC751;&#xB2F5; &#xD504;&#xB85C;&#xD544;&#xC744; &#xD655;&#xC778;&#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC751;&#xB2F5; &#xD504;&#xB85C;&#xD544;&#xACFC; &#xB2E4;&#xB978; &#xB9C8;&#xC6B0;&#xC2A4; Ors&#xB97C; &#xC788;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

S r 1, &#xBE44;&#xC815;&#xC0C1;&#xC801;&#xC73C;&#xB85C; &#xAD11;&#xBC94;&#xC704; &#xD55C; &#xC751;&#xB2F5; &#xD504;&#xB85C;&#xD544;&#xACFC; &#xB9C8;&#xC6B0;&#xC2A4; odorant &#xC218;&#xC6A9; &#xCCB4;.

El modelo de consenso actual en el olfato de mam&#xED;feros es que la detecci&#xF3;n de millones de olores requiere de un gran n&#xFA;mero de receptores olfativos (OR) y que cada uno o interact&#xFA;a de forma selectiva con un peque&#xF1;o subconjunto de sustancias odor&#xED;feras, que suelen ser relacionados en su estructura. En este caso, se reportan las propiedades de respuesta de un olor o que se desv&#xED;a de este modelo: SR1, un rat&#xF3;n o que se expresa abundantemente en las neuronas sensoriales del &#xF3;rgano de comunicaci&#xF3;n interventricular y tambi&#xE9;n del epitelio olfativo principal. Patch-clamp grabaciones revelan que las neuronas olfativas sensoriales (OSN), que expresa SR1 responden a los olores muchos, no relacionados estructuralmente, y en un amplio rango de concentraciones. La mayor&#xED;a de las OSN que expresan un gen locus orientada SR1 que carece de la secuencia de codificaci&#xF3;n SR1 no muestran esta capacidad de respuesta amplia. La transferencia de genes en el sistema de expresi&#xF3;n heter&#xF3;loga Hana3A confirma el perfil de respuesta general de SR1. Puede haber otras ORs rat&#xF3;n con estos perfiles de respuesta generales.

SR1, un receptor odorante del rat&#xF3;n con un perfil de respuesta inusualmente amplio

Tastant detection in the oral cavity involves selective receptors localized at the apical extremity of a subset of specialized taste bud cells called taste receptor cells (TRCs). The identification of the genes coding for the taste receptors involved in this process have greatly improved our understanding of the molecular mechanisms underlying detection. However, how these receptors signal in TRCs, and whether the components of the signaling cascades interact with each other or are organized in complexes is mostly unexplored. Here we report on the identification of three new binding partners for the mouse G protein gamma 13 subunit (Gγ13), a component of the bitter taste receptors signaling cascade. For two of these Gγ13 associated proteins, namely GOPC and MPDZ, we describe the expression in taste bud cells for the first time. Furthermore, we demonstrate by means of a yeast two-hybrid interaction assay that the C terminal PDZ binding motif of Gγ13 interacts with selected PDZ domains in these proteins. In the case of the PDZ domain-containing protein zona occludens-1 (ZO-1), a major component of the tight junction defining the boundary between the apical and baso-lateral region of TRCs, we identified the first PDZ domain as the site of strong interaction with Gγ13. This association was further confirmed by co-immunoprecipitation experiments in HEK 293 cells. In addition, we present immunohistological data supporting partial co-localization of GOPC, MPDZ, or ZO-1, and Gγ13 in taste buds cells. Finally, we extend this observation to olfactory sensory neurons (OSNs), another type of chemosensory cells known to express both ZO-1 and Gγ13. Taken together our results implicate these new interaction partners in the sub-cellular distribution of Gγ13 in olfactory and gustatory primary sensory cells.

Identification of new binding partners of the chemosensory signaling protein Gγ13 expressed in taste and olfactory sensory cells.

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse β2-adrenergic receptor (β2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (β2AR→M71-GFP). By crossing these mice with β2AR→M71-lacZ gene-targeted mice, we find that differentially tagged β2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong β2AR immunoreactivity in dendritic cilia of β2AR→M71-GFP OSNs. These OSNs respond to the β2AR agonist isoproterenol in a dose-dependent manner. Axons of β2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and β2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous β2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of β2AR and β1AR knockout mice. Our findings corroborate the experimental value of the β2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression.

The β2-adrenergic receptor as a surrogate odorant receptor in mouse olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) form the primary elements of the olfactory system. Inserted in the olfactory mucosa lining of the nasal cavity, they are exposed to the environment and their lifespan is brief. Several reports say that OSNs are regularly regenerated during the entire life and that odorant environment affects the olfactory epithelium. However, little is known about the impact of the odorant environment on OSNs at the cellular level and more precisely in the context of early postnatal olfactory exposure. Here we exposed MOR23-green fluorescent protein (GFP) and M71-GFP mice to lyral or acetophenone, ligands for MOR23 or M71, respectively. Daily postnatal exposure to lyral induces plasticity in the population of OSNs expressing MOR23. Their density decreases after odorant exposure, whereas the amount of MOR23 mRNA and protein remain stable in the whole epithelium. Meanwhile, quantitative PCR indicates that each MOR23 neuron has higher levels of olfactory receptor transcripts and also expresses more CNGA2 and phosphodiesterase 1C, fundamental olfactory transduction pathway proteins. Transcript levels return to baseline after 4 weeks recovery. Patch-clamp recordings reveal that exposed MOR23 neurons respond to lyral with higher sensitivity and broader dynamic range while the responses' kinetics were faster. These effects are specific to the odorant-receptor pair lyral-MOR23: there was no effect of acetophenone on MOR23 neurons and no effect of acetophenone and lyral on the M71 population. Together, our results clearly demonstrate that OSNs undergo specific anatomical, molecular, and functional adaptation when chronically exposed to odorants in the early stage of life.

Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR-G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

Xavier Grosmaitre

UMR Centre des Sciences du Goŭt et de l'Alimentation,
CNRS,
INRA,
UMR Centre des Sciences du Goŭt et de l'Alimentation, CNRS, INRA

Université de Bourgogne

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Xavier Grosmaitre

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Université de Bourgogne

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

UMR Centre des Sciences du Goŭt et de l'Alimentation,
CNRS,
INRA,
UMR Centre des Sciences du Goŭt et de l'Alimentation, CNRS, INRA