Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Pinaud, Raphael; Mello, Claudio V.; Velho, Tarciso A. F.; Wynne, Ryan D.; Tremere, Liisa A.

doi:10.1038/nprot.2008.115

Cited by 38 publications

(58 citation statements)

References 30 publications

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“…After injections, animals were stimulated with a medley of conspecific songs. C-E, High-power photomicrographs depicting mRNA expression of zenk (C), c-fos (D), and arc (E), as revealed by fluorescence in situ hybridization (Pinaud et al, 2008b). Importantly, the images shown for vehicle and TMX-treated hemisphere were obtained from the same animal, hybridized in a single reaction batch.…”

Section: Discussionmentioning

confidence: 99%

“…4 A). A fluorescence in situ hybridization protocol developed by our group was used to assess the expression levels of two regulatory and one effector song-inducible IEGs: zenk, c-fos, and arc (Pinaud et al, 2008b). Importantly, we and others previously demonstrated that the activation of the MAPK pathway is necessary for the expression of all three IEGs in the songbird NCM (Cheng and Clayton, 2004;Velho et al, 2005).…”

Section: Local E2 Is Necessary For Mapk-dependent Hearing-driven Genmentioning

confidence: 99%

“…We used a fluorescence in situ hybridization (FISH) protocol that we developed and described in detail previously (Pinaud et al, , 2008b. Briefly, Qiagen miniprep kit was used to purify plasmids containing zebra finch homologs of the zenk, c-fos, and arc genes from bacteria.…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

“…Plasmids were either linearized by digestion with appropriate restriction enzymes (zenk and arc) or amplified by PCR (c-fos). The cDNAs were purified and used as templates for the generation of digoxigenin (DIG)-labeled antisense and sense strand riboprobes, which were purified in G-50 columns and evaluated for yield in a spectrophotometer (Pinaud et al, , 2008b. Sections were sequentially (1) incubated in a 4% paraformaldehyde solution in 0.1 M PBS for 5 min, (2) rinsed in 0.1 M PBS, (3) dehydrated in a standard series of alcohols, (4) air dried, (5) incubated for 10 min at room temperature (RT) in an acetylation solution (1.35% triethanolamine and 0.25% acetic anhydride in water), (6) rinsed three times with 2ϫ saline-sodium phosphate-EDTA (SSPE), and (7) dehydrated in the alcohol series and air dried.…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

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Estradiol Shapes Auditory Processing in the Adult Brain by Regulating Inhibitory Transmission and Plasticity-Associated Gene Expression

Tremere¹,

Jeong²,

Pinaud³

2009

J. Neurosci.

137

219

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Estradiol impacts a wide variety of brain processes, including sex differentiation, mood, and learning. Here we show that estradiol regulates auditory processing of acoustic signals in the vertebrate brain, more specifically in the caudomedial nidopallium (NCM), the songbird analog of the mammalian auditory association cortex. Multielectrode recordings coupled with local pharmacological manipulations in awake animals reveal that both exogenous and locally generated estradiol increase auditory-evoked activity in NCM. This enhancement in neuronal responses is mediated by suppression of local inhibitory transmission. Surprisingly, we also found that estradiol is both necessary and sufficient for the induction of multiple mitogen-activated protein kinase (MAPK)-dependent genes thought to be required for synaptic plasticity and memorization of birdsong. Specifically, we show that local blockade of estrogen receptors or aromatase activity in awake birds decrease song-induced MAPK-dependent gene expression. Infusions of estradiol in acoustically isolated birds induce transcriptional activation of these genes to levels comparable with song-stimulated animals. Our results reveal acute and rapid nongenomic functions for estradiol in central auditory physiology and suggest that such roles may be ubiquitously expressed across sensory systems.

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Section: Discussionmentioning

confidence: 99%

Section: Local E2 Is Necessary For Mapk-dependent Hearing-driven Genmentioning

confidence: 99%

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

See 2 more Smart Citations

Estradiol Shapes Auditory Processing in the Adult Brain by Regulating Inhibitory Transmission and Plasticity-Associated Gene Expression

Tremere¹,

Jeong²,

Pinaud³

2009

J. Neurosci.

137

219

View full text Add to dashboard Cite

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“…Critical troubleshooting steps for optimizing probe hybridization have been described in detail elsewhere (Pinaud et al 2008;Piette et al 2008). Additional troubleshooting recommendations can be found in Table 1.…”

Section: General Commentsmentioning

confidence: 99%

Multi-probe in situ hybridization to whole mount Arabidopsis seedlings

et al. 2011

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In situ RNA-RNA hybridization (ISH) is a molecular method for localization of gene transcripts at the cellular level and is widely used to provide spatial and temporal information regarding gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time, each one observed on separate ISH preparations. Multi-probe whole-mount in situ hybridization is a powerful technique to compare the expression patterns of two or more genes simultaneously in the same tissue or organ. We describe for the first time in plants, the detection of three different mRNAs in a single fixed whole mount Arabidopsis seedling. A combination of bright fluorescent secondary antibodies was used for the detection of riboprobes differentially labeled by digoxigenin, biotin and fluorescein. The 3-D detection of each of the multiple fluorescent hybridization signals or in combination was obtained through confocal laser-scanning microscopy. The reliability of the method was tested in the root, using the PINFORMED (PIN) genes with non-overlapping temporal and spatial expression patterns. In the shoot, a class-I KNOTTED -like homeobox gene from Arabidopsis (KNAT1) with expression restricted to the shoot apical meristem was used in combination with ELONGATOR3 (ELO3) gene. In addition, the expression patterns of ELONGATOR complex gene (ELO2, ELO3) and HISTONE MONOUBIQUITINATION1 (HUB1) genes were analyzed in both shoot and root and a partial overlapping was observed. The whole procedure takes only 6 days.

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Expression and rapid experience‐dependent regulation of type‐A GABAergic receptors in the songbird auditory forebrain

Jeong

Terleph

Burrows

et al. 2011

Developmental Neurobiology

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GABAergic transmission influences sensory processing and experience-dependent plasticity in the adult brain. Little is known about the functional organization of inhibitory circuits in the auditory forebrain of songbirds, a robust model extensively used in the study of central auditory processing of behaviorally relevant communication signals. In particular, no information is currently available on the expression and organization of GABAA receptor-expressing neurons. Here, we studied the distribution and regulation of GABAA receptors in the songbird auditory forebrain, with a specific focus on α5, a subunit implicated in tonic inhibition and sensory learning. We obtained a zebra finch cDNA that encodes the α5-subunit (GABRA5) and carried out a detailed analysis of its expression via in situ hybridization. GABRA5 was highly expressed in the caudomedial nidopallium (NCM), caudomedial mesopallium, and field L2. Using double fluorescence in situ hybridization, we demonstrate that a large fraction of GABRA5-expressing neurons is engaged by auditory experience, as revealed by the song-induced expression of the activity-dependent gene zenk. Remarkably, we also found that α5 expression is rapidly regulated by sensory stimulation: 30 min of conspecific song playbacks significantly increase the number of GABRA5-expressing neurons in NCM, but not in other auditory areas. This effect is selective for α5, but not γ2 transcripts. Our results suggest that α5-containing GABAA receptors likely play a key role in central auditory processing and may contribute to the experience-dependent plasticity underlying auditory learning.

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Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Cited by 38 publications

References 30 publications

Estradiol Shapes Auditory Processing in the Adult Brain by Regulating Inhibitory Transmission and Plasticity-Associated Gene Expression

Estradiol Shapes Auditory Processing in the Adult Brain by Regulating Inhibitory Transmission and Plasticity-Associated Gene Expression

Multi-probe in situ hybridization to whole mount Arabidopsis seedlings

Expression and rapid experience‐dependent regulation of type‐A GABAergic receptors in the songbird auditory forebrain

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