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1.
J Biol Chem ; 290(15): 9767-79, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25713142

RESUMO

Olfaction is one of the most crucial senses for vertebrates regarding foraging and social behavior. Therefore, it is of particular interest to investigate the sense of smell, its function on a molecular level, the signaling proteins involved in the process and the mechanism of required ion transport. In recent years, the precise role of the ion transporter NKCC1 in olfactory sensory neuron (OSN) chloride accumulation has been a controversial subject. NKCC1 is expressed in OSNs and is involved in chloride accumulation of dissociated neurons, but it had not been shown to play a role in mouse odorant sensation. Here, we present electro-olfactogram recordings (EOG) demonstrating that NKCC1-deficient mice exhibit significant defects in perception of a complex odorant mixture (Henkel100) in both air-phase and submerged approaches. Using next generation sequencing (NGS) and RT-PCR experiments of NKCC1-deficient and wild type mouse transcriptomes, we confirmed the absence of a highly expressed ion transporter that could compensate for NKCC1. Additional histological investigations demonstrated a reduced number of cells in the olfactory epithelium (OE), resulting in a thinner neuronal layer. Therefore, we conclude that NKCC1 is an important transporter involved in chloride ion accumulation in the olfactory epithelium, but it is also involved in OSN neurogenesis.


Assuntos
Cloretos/metabolismo , Neurogênese/genética , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Animais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Immunoblotting , Transporte de Íons/genética , Masculino , Camundongos Endogâmicos , Camundongos Knockout , Microscopia Confocal , Odorantes , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Olfato , Membro 2 da Família 12 de Carreador de Soluto/deficiência , Transcriptoma
2.
PLoS One ; 11(8): e0159640, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27494699

RESUMO

The influence of the sex steroid hormones progesterone and estradiol on physiology and behavior during menstrual cycles and pregnancy is well known. Several studies indicate that olfactory performance changes with cyclically fluctuating steroid hormone levels in females. Knowledge of the exact mechanisms behind how female sex steroids modulate olfactory signaling is limited. A number of different known genomic and non-genomic actions that are mediated by progesterone and estradiol via interactions with different receptors may be responsible for this modulation. Next generation sequencing-based RNA-Seq transcriptome data from the murine olfactory epithelium (OE) and olfactory receptor neurons (ORNs) revealed the expression of several membrane progestin receptors and the estradiol receptor Gpr30. These receptors are known to mediate rapid non-genomic effects through interactions with G proteins. RT-PCR and immunohistochemical staining results provide evidence for progestin and estradiol receptors in the ORNs. These data support the hypothesis that steroid hormones are capable of modulating the odorant-evoked activity of ORNs. Here, we validated this hypothesis through the investigation of steroid hormone effects by submerged electro-olfactogram and whole cell patch-clamp recordings of ORNs. For the first time, we demonstrate that the sex steroid hormones progesterone and estradiol decrease odorant-evoked signals in the OE and ORNs of mice at low nanomolar concentrations. Thus, both of these sex steroids can rapidly modulate the odor responsiveness of ORNs through membrane progestin receptors and the estradiol receptor Gpr30.


Assuntos
Estradiol/farmacologia , Potenciais Evocados/efeitos dos fármacos , Neurônios Receptores Olfatórios/metabolismo , Progesterona/farmacologia , Animais , Benzaldeídos/farmacologia , AMP Cíclico/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos , Técnicas de Patch-Clamp , RNA/química , RNA/genética , RNA/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Análise de Sequência de RNA
3.
PLoS One ; 10(1): e0113170, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25590618

RESUMO

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.


Assuntos
Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Transcriptoma/fisiologia , Animais , Feminino , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Transgênicos , Condutos Olfatórios/metabolismo , Transdução de Sinais/fisiologia
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