Nonradioactive In Situ Hybridization in Combination with Tract-Tracing

Stornetta, Ruth L.

doi:10.1007/0-387-28942-9_8

Cited by 6 publications

(5 citation statements)

References 51 publications

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“…This discrepancy is attributed to the observation that the in situ hybridization procedure, albeit compatible with immunohistochemistry, challenges subsequent immunocytochemical procedures (Stornetta and Guyenet, 2006;Pérez-Manso et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Origin of calretinin‐containing, vesicular glutamate transporter 2‐coexpressing fiber terminals in the entorhinal cortex of the rat

Wouterlood

Aliane

Boekel

et al. 2007

J of Comparative Neurology

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The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.

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Section: Discussionmentioning

confidence: 99%

Origin of calretinin‐containing, vesicular glutamate transporter 2‐coexpressing fiber terminals in the entorhinal cortex of the rat

Wouterlood

Aliane

Boekel

et al. 2007

J of Comparative Neurology

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“…The major player in terms of markers for glutamatergic transmission in respiratory-related neurons is VGlut2, since in brainstem, VGlut1 is located predominantly in neurons supplying mossy fibers to the cerebellum (Hisano et al, 2002; Stornetta and Guyenet, 2005) and VGlut3 is limited to neurons in raphe magnus, parapyramidal raphe and dorsal and median raphe nuclei (Herzog et al, 2004; Nakamura et al, 2004; Stornetta et al, 2005) and in nitroxidergic neurons in the nucleus of the solitary tract (NTS) (Lin and Talman, 2005). The ISH method for using non-radioactive cRNA probes for GAD 65/67, GlyT2 or VGlut2 is performed using free-floating sections from paraformaldehyde fixed brains.…”

Section: Introductionmentioning

confidence: 99%

“…These probes can label neuronal somata with high signal-to-noise ratios and the labeling can be combined with other immunocytochemical and tract-tracing methods (c.f. (Stornetta et al, 2001, 2002a, 2004; Stornetta and Guyenet, 2005)).…”

Section: Introductionmentioning

confidence: 99%

Identification of neurotransmitters and co-localization of transmitters in brainstem respiratory neurons

Stornetta

2008

Respiratory Physiology & Neurobiology

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Identifying the major ionotropic neurotransmitter in a respiratory neuron is of critical importance in determining how the neuron fits into the respiratory system, whether in producing or modifying respiratory drive and rhythm. There are now several groups of respiratory neurons whose major neurotransmitters have been identified and in some of these cases, more than one transmitter have been identified in particular neurons. This review will describe the physiologically identified neurons in major respiratory areas that have been phenotyped for major ionotropic transmitters as well as those where more than one transmitter has been identified. Although the purpose of the additional transmitter has not been elucidated for any of the respiratory neurons, some examples from other systems will be discussed.

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“…When considering the central nervous system, such combinations have been successfully applied to both the developing brain (Pott and Fuss, 1996) and the brainstem of adult rats (Stornetta et al, 2002a). In general, the simultaneous hybridization using biotin-and digoxigenin-labeled riboprobes is undoubtedly the most widely employed, irrespective of the method of visualization chosen (for review, see Chevalier et al, 1997;Le Moine et al, 1995;O'Neill and Bier, 1994;Stornetta and Guyenet, 2006).…”

Section: Selection Of the Most Appropriate Combination Of Labeled Promentioning

confidence: 98%

“…In order to avoid combining the use of two protocols of a different nature (radioactive and non-radioactive), several procedures for dual ISH using non-radiolabeled oligoprobes alone are available in which digoxigenin-and biotin-labeled riboprobes are used to simultaneously visualize two different transcripts (for review, see Chevalier et al, 1997;Le Moine et al, 1995;O'Neill and Bier, 1994;Stornetta and Guyenet, 2006). The use of fluorescein as an alternative to biotin for riboprobe labeling also provides nice results (Clay and Ramakrishnan, 2005;Hansen et al, 2000;Hauptmann, 2001;Hopman et al, 1997;Ivanova et al, 2004;Jezzini et al, 2005;Kapsimali et al, 2001;Pott and Fuss, 1996;Yasuda, 2004, 2006).…”

Section: Introductionmentioning

confidence: 99%

Detection of two different mRNAs in a single section by dual in situ hybridization: A comparison between colorimetric and fluorescent detection

Barroso‐Chinea

Aymerich

Castle

et al. 2007

Journal of Neuroscience Methods

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Nonradioactive In Situ Hybridization in Combination with Tract-Tracing

Cited by 6 publications

References 51 publications

Origin of calretinin‐containing, vesicular glutamate transporter 2‐coexpressing fiber terminals in the entorhinal cortex of the rat

Origin of calretinin‐containing, vesicular glutamate transporter 2‐coexpressing fiber terminals in the entorhinal cortex of the rat

Identification of neurotransmitters and co-localization of transmitters in brainstem respiratory neurons

Detection of two different mRNAs in a single section by dual in situ hybridization: A comparison between colorimetric and fluorescent detection

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