Specific identification of cardiac vagal afferent neurons in culture

M. Bitticaca, Helen E Raybould, H. R. Middlekauff

Research output: Contribution to journalArticle

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Abstract

Introduction Cardiac vagal afferent nerves are sensitive to mechanical and chemical stimuli and regulate systemic reflexes. The soma of these neurons reside in the nodose ganglia (NG)-a heterogenous population of visceral neurons. It would be desirable to identify in vitro cardiac specific neurons in the NG enabling functional neuronal studies. The purpose of this study is to identify in culture a subpopulation of cardiac specific neurons in the NG using retrograde labeling with the fluorescent dextran conjugate Fluoro-ruby (FR 10%). Methods 11 adult male Sprague-Dawley rats were used. 4×4ul injections were made into the base of the L and R ventricles. As controls, 16ul of FR was injected into the thoracic cavity (n=2) or intra-arterially (n=2). A fifth control underwent L cervical vagotomy at the time of cardiac injections. After a survival time of 2-3 weeks, animals were euthanized and tissues were fixed in 4% PF, sectioned (10um) and thaw mounted for visualization of FR using a microscope equipped with epifluorescence. Sections were examined by two investigators blinded to the experimental protocol. Positive cells were prospectively defined as cells with heavy granular labeling with visible nuclei. Results There was 100% correlation between the investigators: positive cells were present bilaterally without specific distribution in sections from 5/6 rats. The number of labeled cells ranged from 0-4 cells per section. The diameter of 83% of labeled neurons was 20um and 17% of cells≥30um. In control rats no heavy granular labeled cells were identified. After the left vagotomy, labeled cells were present only in the right NG. In cultures of NG neurons, FR containing cells were present and viable. Conclusion With this nontoxic fluorescent dye technique, living vagal afferents supplying the heart can be identified specifically. This methodology will facilitate functional studies of this target population under physiologic and pathophysiological conditions.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number3
StatePublished - 1997
Externally publishedYes

Fingerprint

Afferent Neurons
sensory neurons
Nodose Ganglion
Neurons
neurons
cells
Vagotomy
Labeling
Rats
Cells
Rat control
Research Personnel
rats
Thoracic Cavity
Carisoprodol
Injections
injection
Dextrans
thoracic cavity
Fluorescent Dyes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Specific identification of cardiac vagal afferent neurons in culture. / Bitticaca, M.; Raybould, Helen E; Middlekauff, H. R.

In: FASEB Journal, Vol. 11, No. 3, 1997.

Research output: Contribution to journalArticle

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abstract = "Introduction Cardiac vagal afferent nerves are sensitive to mechanical and chemical stimuli and regulate systemic reflexes. The soma of these neurons reside in the nodose ganglia (NG)-a heterogenous population of visceral neurons. It would be desirable to identify in vitro cardiac specific neurons in the NG enabling functional neuronal studies. The purpose of this study is to identify in culture a subpopulation of cardiac specific neurons in the NG using retrograde labeling with the fluorescent dextran conjugate Fluoro-ruby (FR 10{\%}). Methods 11 adult male Sprague-Dawley rats were used. 4×4ul injections were made into the base of the L and R ventricles. As controls, 16ul of FR was injected into the thoracic cavity (n=2) or intra-arterially (n=2). A fifth control underwent L cervical vagotomy at the time of cardiac injections. After a survival time of 2-3 weeks, animals were euthanized and tissues were fixed in 4{\%} PF, sectioned (10um) and thaw mounted for visualization of FR using a microscope equipped with epifluorescence. Sections were examined by two investigators blinded to the experimental protocol. Positive cells were prospectively defined as cells with heavy granular labeling with visible nuclei. Results There was 100{\%} correlation between the investigators: positive cells were present bilaterally without specific distribution in sections from 5/6 rats. The number of labeled cells ranged from 0-4 cells per section. The diameter of 83{\%} of labeled neurons was 20um and 17{\%} of cells≥30um. In control rats no heavy granular labeled cells were identified. After the left vagotomy, labeled cells were present only in the right NG. In cultures of NG neurons, FR containing cells were present and viable. Conclusion With this nontoxic fluorescent dye technique, living vagal afferents supplying the heart can be identified specifically. This methodology will facilitate functional studies of this target population under physiologic and pathophysiological conditions.",
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