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1.
Chem Senses ; 44(8): 583-592, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31420672

RESUMO

Published evidence suggests that inherent rhythmically active or "bursting" primary olfactory receptor neurons (bORNs) in crustaceans have the previously undescribed functional property of encoding olfactory information by having their rhythmicity entrained by the odor stimulus. In order to determine whether such bORN-based encoding is a fundamental feature of olfaction that extends beyond crustaceans, we patch-clamped bORN-like ORNs in mice, characterized their dynamic properties, and show they align with the dynamic properties of lobster bORNs. We then characterized bORN-like activity by imaging the olfactory epithelium of OMP-GCaMP6f mice. Next, we showed rhythmic activity is not dependent upon the endogenous OR by patching ORNs in OR/GFP mice. Lastly, we showed the properties of bORN-like ORNs characterized in mice generalize to rats. Our findings suggest encoding odor time should be viewed as a fundamental feature of olfaction with the potential to be used to navigate odor plumes in animals as diverse as crustaceans and mammals.

2.
J Cell Sci ; 132(5)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30665891

RESUMO

Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.

3.
J Neurosci ; 38(34): 7462-7475, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30061191

RESUMO

Cilia of olfactory sensory neurons (OSNs) are the primary site of odor binding; hence, their loss results in anosmia, a clinical manifestation of pleiotropic ciliopathies for which there are no curative therapies. We used OSN-specific Ift88 knock-out mice (Ift88osnKO) of both sexes to examine the mechanisms of ciliopathy-induced olfactory dysfunction and the potential for gene replacement to rescue odorant detection, restore olfactory circuitry, and restore odor-guided behaviors. Loss of OSN cilia in Ift88osnKO mice resulted in substantially reduced odor detection and odor-driven synaptic activity in the olfactory bulb (OB). Defects in OSN axon targeting to the OB were also observed in parallel with aberrant odor-guided behavior. Intranasal gene delivery of wild-type IFT88 to Ift88osnKO mice rescued OSN ciliation and peripheral olfactory function. Importantly, this recovery of sensory input in a limited number of mature OSNs was sufficient to restore axonal targeting in the OB of juvenile mice, and with delayed onset in adult mice. In addition, restoration of sensory input re-established course odor-guided behaviors. These findings highlight the spare capacity of the olfactory epithelium and the plasticity of primary synaptic input into the central olfactory system. The restoration of peripheral and central neuronal function supports the potential for treatment of ciliopathy-related anosmia using gene therapy.SIGNIFICANCE STATEMENT Ciliopathies, for which there are no curative therapies, are genetic disorders that alter cilia morphology and/or function in numerous tissue types, including the olfactory system, leading to sensory dysfunction. We show that in vivo intranasal gene delivery restores peripheral olfactory function in a ciliopathy mouse model, including axonal targeting in the juvenile and adult olfactory bulb. Gene therapy also demonstrated restoration of olfactory perception by rescuing odor-guided behaviors. Understanding the therapeutic window and viability for gene therapy to restore odor detection and perception may facilitate translation of therapies to ciliopathy patients with olfactory dysfunctions.


Assuntos
Ciliopatias/terapia , Terapia Genética , Transtornos do Olfato/terapia , Neurônios Receptores Olfatórios/fisiologia , Proteínas Supressoras de Tumor/uso terapêutico , Adenoviridae , Administração Intranasal , Fatores Etários , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Cílios/ultraestrutura , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Odorantes , Bulbo Olfatório/fisiopatologia , Mucosa Olfatória/patologia , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia
4.
Mol Ther ; 25(4): 904-916, 2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28237838

RESUMO

Olfactory dysfunction is a pervasive but underappreciated health concern that affects personal safety and quality of life. Patients with olfactory dysfunctions have limited therapeutic options, particularly those involving congenital diseases. Bardet-Biedl syndrome (BBS) is one such disorder, where olfactory loss and other symptoms manifest from defective cilium morphology and/or function in various cell types/tissues. Olfactory sensory neurons (OSNs) of BBS mutant mice lack the capacity to build/maintain cilia, rendering the cells incapable of odor detection. Here we examined OSN cilium defects in Bbs1 mutant mice and assessed the utility of gene therapy to restore ciliation and function in young and adult mice. Bbs1 mutant mice possessed short residual OSN cilia in which BBSome protein trafficking and odorant detection were defective. Gene therapy with an adenovirus-delivered wild-type Bbs1 gene restored OSN ciliation, corrected BBSome cilium trafficking defects, and returned acute odor responses. Finally, using clinically approved AAV serotypes, we demonstrate, for the first time, the capacity of AAVs to restore ciliation and odor detection in OSNs of Bbs1 mutants. Together, our data demonstrate that OSN ciliogenesis can be promoted in differentiated cells of young and adult Bbs1 mutants and highlight the potential of gene therapy as a viable restorative treatment for congenital olfactory disorders.


Assuntos
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/fisiopatologia , Terapia Genética , Neurônios Receptores Olfatórios/metabolismo , Alelos , Animais , Síndrome de Bardet-Biedl/terapia , Cílios/metabolismo , Cílios/patologia , Dependovirus/genética , Modelos Animais de Doenças , Expressão Ectópica do Gene , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Percepção Olfatória/genética , Fenótipo , Transporte Proteico , Transdução Genética
5.
Front Cell Neurosci ; 10: 97, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27147969

RESUMO

Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs.

6.
PLoS One ; 8(4): e60551, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23573266

RESUMO

The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.


Assuntos
Proteínas de Artrópodes/metabolismo , Dendritos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Palinuridae/fisiologia , Receptores Ionotrópicos de Glutamato/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Expressão Gênica , Dados de Sequência Molecular , Neurônios Receptores Olfatórios/ultraestrutura , Especificidade de Órgãos , Palinuridae/citologia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/genética , Homologia de Sequência de Aminoácidos , Olfato
7.
PLoS One ; 8(4): e61553, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23585911

RESUMO

Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs.


Assuntos
Odorantes/análise , Neurônios Receptores Olfatórios/fisiologia , Receptores Odorantes/metabolismo , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Feminino , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Microscopia de Fluorescência , Morfolinas/farmacologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Pirimidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazolidinedionas/farmacologia
8.
PLoS One ; 7(9): e46358, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23050020

RESUMO

Peptide hormones and their cognate receptors belonging to neuropeptide Y (NPY) family mediate diverse biological functions in a number of tissues. Recently, we discovered the presence of the gut satiation peptide YY (PYY) in saliva of mice and humans and defined its role in the regulation of food intake and body weight maintenance. Here we report the systematic analysis of expression patterns of all NPY receptors (Rs), Y1R, Y2R, Y4R, and Y5R in lingual epithelia in mice. Using four independent assays, immunohistochemistry, in situ hybridization, immunocytochemistry and RT PCR, we show that the morphologically different layers of the keratinized stratified epithelium of the dorsal layer of the tongue express Y receptors in a very distinctive yet overlapping pattern. In particular, the monolayer of basal progenitor cells expresses both Y1 and Y2 receptors. Y1Rs are present in the parabasal prickle cell layer and the granular layer, while differentiated keratinocytes display abundant Y5Rs. Y4Rs are expressed substantially in the neuronal fibers innervating the lamina propria and mechanoreceptors. Basal epithelial cells positive for Y2Rs respond robustly to PYY(3-36) by increasing intracellular Ca(2+) suggesting their possible functional interaction with salivary PYY. In taste buds of the circumvallate papillae, some taste receptor cells (TRCs) express YRs localized primarily at the apical domain, indicative of their potential role in taste perception. Some of the YR-positive TRCs are co-localized with neuronal cell adhesion molecule (NCAM), suggesting that these TRCs may have synaptic contacts with nerve terminals. In summary, we show that all YRs are abundantly expressed in multiple lingual cell types, including epithelial progenitors, keratinocytes, neuronal dendrites and TRCs. These results suggest that these receptors may be involved in the mediation of a wide variety of functions, including proliferation, differentiation, motility, taste perception and satiation.


Assuntos
Epitélio/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Língua/citologia , Animais , Linhagem Celular , Humanos , Hibridização In Situ , Técnicas In Vitro , Camundongos , Receptores de Neuropeptídeo Y/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
PLoS One ; 7(4): e34843, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22514675

RESUMO

An emerging idea in olfaction is that temporal coding of odor specificity can be intrinsic to the primary olfactory receptor neurons (ORNs). As a first step towards understanding whether lobster ORNs are capable of generating odor-specific temporal activity and what mechanisms underlie any such heterogeneity in discharge pattern, we characterized different patterns of activity in lobster ORNs individually and ensemble using patch-clamp recording and calcium imaging. We demonstrate that lobster ORNs show tonic excitation, tonic inhibition, phaso-tonic excitation, and bursting, and that these patterns are faithfully reflected in the calcium signal. We then demonstrate that the various dynamic patterns of response are inherent in the cells, and that this inherent heterogeneity is largely determined by heterogeneity in the underlying intrinsic conductances.


Assuntos
Neurônios Receptores Olfatórios/metabolismo , Animais , Cálcio/metabolismo , Eletrofisiologia , Neurônios Receptores Olfatórios/fisiologia
10.
J Neurosci ; 31(1): 273-80, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21209212

RESUMO

Phosphoinositide signaling, in particular, phosphoinositide 3-kinase (PI3K) signaling, has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand this phenomenon we investigated PI3K-dependent inhibition between single odorant pairs. The concentration-dependent inhibition of the response of native rat ORNs to octanol by citral is PI3K dependent; blocking PI3K activity with the ß and γ isoform-specific inhibitors AS252424 (5-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-dione) and TGX221(7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido [1,2-a]pyrimidin-4-one) eliminated or strongly reduced the inhibition. Interestingly, blocking PI3K also changed the apparent agonist strength of the otherwise noncompetitive antagonist citral. The excitation evoked by citral after blocking PI3K, could be suppressed by the adenylate cyclase III (ACIII) blockers MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride) and SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine], indicating that citral could also activate ACIII, presumably through the canonical olfactory receptor (OR). The G-protein G(ß)γ subunit blockers suramin (8,8'-[carbonylbis[imino-3,1-phenylen ecarbonylimino(4-methyl-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalenetrisulfonic acid), gallein (3',4',5',6'-tetrahydroxyspiro[isobenzofuran-1(3H),9'-(9H)xanthen]-3-one), and M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9CI)]) suppressed citral's inhibition of the response to octanol, indicating that the activation of PI3K by citral was G-protein dependent, consistent with the idea that inhibition acts via the canonical OR. Lilial similarly antagonized the response to isoamyl acetate in other ORNs, indicating the effect generalizes to at least one other odorant pair. The ability of methyl-isoeugenol, limonene, α-pinene, isovaleric acid, and isosafrole to inhibit the response of other ORNs to IBMX (3-isobutyl-1-methylxanthine)/forskolin in a PI3K-dependent manner argues the effect generalizes to yet other structurally dissimilar odorants. Our findings collectively raise the interesting possibility that the OR serves as a molecular logic gate when mammalian ORNs are activated by natural, complex mixtures containing both excitatory and inhibitory odorants.


Assuntos
Neurônios Receptores Olfatórios/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Cicloexanos/farmacologia , Relação Dose-Resposta a Droga , Interações de Medicamentos , Inibidores Enzimáticos/farmacologia , Modelos Biológicos , Monoterpenos/farmacologia , Odorantes , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Suramina/farmacologia , Xantenos/farmacologia
11.
J Neurophysiol ; 103(2): 1114-22, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20032232

RESUMO

Odorants inhibit as well as excite olfactory receptor neurons (ORNs) in many species of animals. Cyclic nucleotide-dependent activation of canonical mammalian ORNs is well established but it is still unclear how odorants inhibit these cells. Here we further implicate phosphoinositide-3-kinase (PI3K), an indispensable element of PI signaling in many cellular processes, in olfactory transduction in rodent ORNs. We show that odorants rapidly and transiently activate PI3K in the olfactory cilia and in the olfactory epithelium in vitro. We implicate known G-protein-coupled isoforms of PI3K and show that they modulate not only the magnitude but also the onset kinetics of the electrophysiological response of ORNs to complex odorants. Finally, we show that the ability of a single odorant to inhibit another can be PI3K dependent. Our collective results provide compelling support for the idea that PI3K-dependent signaling mediates inhibitory odorant input to mammalian ORNs and at least in part contributes to the mixture suppression typically seen in the response of ORNs to complex natural odorants.


Assuntos
Odorantes , Neurônios Receptores Olfatórios/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Ratos , Ratos Sprague-Dawley
12.
J Neurophysiol ; 98(4): 2357-69, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17715188

RESUMO

Sensory neurons in the mouse vomeronasal organ consist of two major groups, apical and basal, that project to different brain regions, express unique sets of receptors, and serve distinct functions. Electrical properties of these two subpopulations, however, have not been systematically characterized. V1rb2-tau-GFP and V2r1b-tau-GFP tagged vomeronasal sensory neurons (VSNs) were selected as prototypical apical or basal VSNs, respectively, and their biophysical properties were analyzed in acute slices that minimized cell damage. Basal V2r1b-expressing VSNs had voltage-gated conductances, and especially Na(+) (Nav) and Ca(2+) (Cav) currents, that were substantially larger than those observed in apical V1rb2 VSNs, although the resting membrane potential, input resistance, and membrane capacitance were similar in both cell types. Of several types of Cav currents, T-type and L-type Cav currents contributed to action potential firing, and both currents alone were capable of generating oscillatory Ca(2+) spikes. The L-type Cav current was uniquely coupled to a BK large-conductance K(+) current, and interplay between these channels played a critical role in repolarizing spikes and maintaining persistent firing in VSNs. Larger Nav and Cav conductances, along with a more positive inactivation voltage of the Nav current in the V2r1b VSNs, contributed to the larger spike amplitude and higher spike frequency induced by depolarizing current in these cells compared with V1rb2 VSNs. Basal GFP-negative VSNs and V2r1b VSNs responded to prolonged depolarization with persistent, but adapting discharge that could be relevant in sensory adaptation. Collectively, these results suggest a novel mechanism for regulating and encoding neuronal activity in the accessory olfactory system.


Assuntos
Canais Iônicos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores de Vasopressinas/fisiologia , Órgão Vomeronasal/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Interpretação Estatística de Dados , Eletrofisiologia , Proteínas de Fluorescência Verde , Técnicas In Vitro , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Camundongos Transgênicos , Condutos Olfatórios/fisiologia , Técnicas de Patch-Clamp , Receptores de Vasopressinas/genética , Canais de Sódio/genética , Canais de Sódio/fisiologia , Órgão Vomeronasal/citologia , Órgão Vomeronasal/metabolismo
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