Genetic Tracing of Nav1.8-Expressing Vagal Afferents in the Mouse

Division of Hypothalamic Research, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
The Journal of Comparative Neurology (Impact Factor: 3.23). 10/2011; 519(15):3085-101. DOI: 10.1002/cne.22667
Source: PubMed


Nav1.8 is a tetrodotoxin-resistant sodium channel present in large subsets of peripheral sensory neurons, including both spinal and vagal afferents. In spinal afferents, Nav1.8 plays a key role in signaling different types of pain. Little is known, however, about the exact identity and role of Nav1.8-expressing vagal neurons. Here we generated mice with restricted expression of tdTomato fluorescent protein in all Nav1.8-expressing afferent neurons. As a result, intense fluorescence was visible in the cell bodies, central relays, and sensory endings of these neurons, revealing the full extent of their innervation sites in thoracic and abdominal viscera. For instance, vagal and spinal Nav1.8-expressing endings were seen clearly within the gastrointestinal mucosa and myenteric plexus, respectively. In the gastrointestinal muscle wall, labeled endings included a small subset of vagal tension receptors but not any stretch receptors. We also examined the detailed innervation of key metabolic tissues such as liver and pancreas and evaluated the anatomical relationship of Nav1.8-expressing vagal afferents with select enteroendocrine cells (i.e., ghrelin, glucagon, GLP-1). Specifically, our data revealed the presence of Nav1.8-expressing vagal afferents in several metabolic tissues and varying degrees of proximity between Nav1.8-expressing mucosal afferents and enteroendocrine cells, including apparent neuroendocrine apposition. In summary, this study demonstrates the power and versatility of the Cre-LoxP technology to trace identified visceral afferents, and our data suggest a previously unrecognized role for Nav1.8-expressing vagal neurons in gastrointestinal functions.

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    • "VAN are well known to carry the bulk of the information about the nutritional content of a meal from the gastrointestinal tract to the brain, and lead to meal termination [24]. Although we did not specifically study LepR expression in VAN innervating the gastrointestinal tract, Nav1.8-cre mice have been demonstrated to have extensive vagal innervation of the gastrointestinal tract [54]. Furthermore , retrograde tracing experiments have established that VAN innervating the gut are located in the caudal portion [55] and express CCK1 receptor [56]. "
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    ABSTRACT: The vagal afferent pathway senses hormones released from the gut in response to nutritional cues and relays these signals to the brain. We tested the hypothesis that leptin resistance in vagal afferent neurons (VAN) is responsible for the onset of hyperphagia by developing a novel conditional knockout mouse to delete leptin receptor selectively in sensory neurons (Nav1.8/LepRfl/fl mice). Chow fed Nav1.8/LepRfl/fl mice weighed significantly more and had increased adiposity compared with wildtype mice. Cumulative food intake, meal size, and meal duration in the dark phase were increased in Nav1.8/LepRfl/fl mice; energy expenditure was unaltered. Reduced satiation in Nav1.8/LepRfl/fl mice is in part due to reduced sensitivity of VAN to CCK and the subsequent loss of VAN plasticity. Crucially Nav1.8/LepRl/fl mice did not gain further weight in response to a high fat diet. We conclude that disruption of leptin signaling in VAN is sufficient and necessary to promote hyperphagia and obesity.
    Full-text · Article · Sep 2014 · Molecular Metabolism
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    • "so that all Nav1.8-positive neurons are labelled [22]. Example sections of dorsal root ganglia (DRG) from Nav1.8Tomato mice at the 4th lumbar spinal level, which innervate the hindpaw (L4 - Figure 2a) contain proportionally less Nav1.8-positive "
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    ABSTRACT: Transgenic mouse behavioural analysis has furthered our understanding of the molecular and cellular mechanisms underlying damage sensing and pain. However, it is not unusual for conflicting data on the pain phenotypes of knockout mice to be generated by reputable groups. Here we focus on some technical aspects of measuring mouse pain behaviour that are often overlooked, which may help explain discrepancies in the pain literature. We examined touch perception using von Frey hairs and mechanical pain thresholds using the Randall-Selitto test. Thermal pain thresholds were measured using the Hargreaves apparatus and a thermal place preference test. Sodium channel Nav1.7 knockout mice show a mechanical deficit in the hairy skin, but not the paw, whilst shaving the abdominal hair abolished this phenotype. Nav1.7, Nav1.8 and Nav1.9 knockout mice show deficits in noxious mechanosensation in the tail, but not the paw. TRPA1 knockout mice, however, have a loss of noxious mechanosensation in the paw but not the tail. Studies of heat and cold sensitivity also show variability depending on the intensity of the stimulus. Deleting Nav1.7, Nav1.8 or Nav1.9 in Nav1.8-positive sensory neurons attenuates responses to slow noxious heat ramps, whilst responses to fast noxious heat ramps are only reduced when Nav1.7 is lost in large diameter sensory neurons. Deleting Nav1.7 from all sensory neurons attenuates responses to noxious cooling but not extreme cold. Finally, circadian rhythms dramatically influence behavioural outcome measures such as von Frey responses, which change by 80% over the day. These observations demonstrate that fully characterising the phenotype of a transgenic mouse strain requires a range of behavioural pain models. Failure to conduct behavioural tests at different anatomical locations, stimulus intensities, and at different points in the circadian cycle may lead to a pain behavioural phenotype being misinterpreted, or missed altogether.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "vector delivery. Administration of lower doses of AAV9 vectors in the cisterna magna rather that the lumbar intrathecal space may achieve preferential gene transfer to nodose compared to spinal ganglia, providing an alternative approach for the study of the vagal sensory innervation of peripheral organs (Kollarik et al., 2010; Gautron et al., 2011). The low level of GFP-ir in the solitary tract after i.v. "
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    ABSTRACT: Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation.
    Full-text · Article · Jun 2014 · Frontiers in Neuroanatomy
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