RESUMO
In the vertebrate olfactory tract new neurons are continuously produced throughout life. It is widely believed that neurogenesis contributes to learning and memory and can be regulated by immune signaling molecules. Proteins originally identified in the immune system have subsequently been localized to the developing and adult nervous system. Previously, we have shown that olfactory imprinting, a specific type of long-term memory, is correlated with a transcriptional response in the olfactory organs that include up-regulation of genes associated with the immune system. To better understand the immune architecture of the olfactory organs we made use of cell-specific fluorescent reporter lines in dissected, intact adult brains of zebrafish to examine the association of the olfactory sensory neurons with neutrophils and blood-lymphatic vasculature. Surprisingly, the olfactory organs contained the only neutrophil populations observed in the brain; these neutrophils were localized in the neural epithelia and were associated with the extensive blood vasculature of the olfactory organs. Damage to the olfactory epithelia resulted in a rapid increase of neutrophils both within the olfactory organs as well as the central nervous system. Analysis of cell division during and after damage showed an increase in BrdU labeling in the neural epithelia and a subset of the neutrophils. Our results reveal a unique population of neutrophils in the olfactory organs that are associated with both the olfactory epithelia and the lymphatic vasculature suggesting a dual olfactory-immune function for this unique sensory system.
Assuntos
Neutrófilos , Neurônios Receptores Olfatórios , Animais , Bulbo Olfatório , Mucosa Olfatória , Neurônios Receptores Olfatórios/metabolismo , Peixe-ZebraRESUMO
CD36 scavenger receptor is expressed in a subpopulation of olfactory sensory neurons (OSNs). These neurons express canonical olfactory signaling machinery; however, not all odorant receptors (ORs) are coexpressed with CD36. In situ hybridization (ISH) enables the detection of nucleic acids in tissues, cells, or isolated chromosomes. The development of nonradioactive and stable labeling probes almost 30 years ago, allowed to routinely perform this technique employing different labeling strategies in one experiment. ISH is widely used in the field of neurobiology of the sense of smell, providing valuable neuroanatomical information regarding the molecular organization of the olfactory epithelium (OE). Here we show a method for studying CD36+-OSNs and provide a detailed protocol for chromogenic ISH, one- or two-color fluorescent ISH, which can be combined with immunofluorescence and are suitable for Cd36 mRNA probing simultaneous to other transcripts and/or proteins labeling.
Assuntos
Antígenos CD36/metabolismo , Hibridização In Situ/métodos , Mucosa Olfatória/metabolismo , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Animais , Antígenos CD36/genética , Humanos , Mucosa Olfatória/citologia , RNA Mensageiro/genéticaRESUMO
The mouse olfactory epithelium is composed of a heterogeneous population of olfactory sensory neurons, where each neuron expresses one single type of odorant receptor gene, out of a repertoire of ~1000 different genes. Fluorescent-activated cell sorting (FACS) is a powerful technique, which can be used to isolate a cellular subpopulation from a heterogeneous tissue. The sorted neurons can then be used in gene expression studies, or analyzed for the presence of different DNA epigenetic modification marks. Here we describe a method to separate a subpopulation of olfactory sensory neurons expressing the odorant receptor Olfr17. In this method, the main olfactory epithelium from transgenic Olfr17-IRES-GFP mice is dissociated into single cells, followed by separation of the GFP positive cells by FACS.
Assuntos
Citometria de Fluxo/métodos , Mucosa Olfatória/citologia , Receptores Odorantes , Células Receptoras Sensoriais/citologia , Animais , Camundongos , Camundongos Transgênicos , Mucosa Olfatória/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
Olfactory perception plays an important role in food flavor. Humans have around 400 odorant receptors (ORs), which can be activated by an enormous number of odorants in a combinatorial fashion. To date, only a few odorant receptors have been linked to their respective odorants, due to the difficulties in expressing these receptor proteins in heterologous cell systems. In vivo approaches allow for the analysis of odorant-receptor interactions in their native environment and have the advantage that the complete OR repertoire is simultaneously tested. Once mouse odorant-receptor pairs are defined, one can search for the corresponding human orthologues, which can be validated against the odorants in heterologous cells. Thus, the combination of in vivo and in vitro methods should contribute to the identification of human ORs that recognize odorants of interest, such as key food odorants.