an-effective-manual-deboning-method-to-prepare-intact-mouse-nasal

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

University of Maryland Baltimore County

Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, but D-tubocurarine did not. U73122, a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+ stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.

Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors.

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+ influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+ responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+ store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.

Taste receptor cell responses to the bitter stimulus denatonium involve Ca2+ influx via store-operated channels.

Sour taste is mediated by acids with the degree of sourness a function of proton concentration. Recently, several members of the acid-sensing ion channel subfamily (ASICs) were cloned from taste cells and proposed to mediate sour taste. However, it is not known whether sour responses in taste cells resemble the responses mediated by ASICs. Using the whole cell patch-clamp technique and Na(+) imaging, we have characterized responses to acid stimuli in isolated rat vallate taste cells. Citric acid (pH 5) induced a large, rapidly activating inward current in most taste cells tested. The response showed various degrees of desensitization with prolonged stimulation. Current amplitudes were pH dependent, and adapting with acidic bath solutions reduced subsequent responses to acid stimulation. Amiloride (100-500 microM) partially and reversibly suppressed the acid-induced current. The current-voltage relationship showed reversal potential near the Na(+) equilibrium potential, suggesting that the current is carried predominantly by Na(+). These data were consistent with Na(+) imaging experiments showing that acid stimulation resulted in increases in intracellular Na(+). Taken together, these data indicate that acid-induced currents in vallate taste cells share general properties with ASICs expressed in heterologous cells and sensory neurons that express ASIC subunits. The large amplitude of the current and its existence in a high percentage of taste cells imply that ASICs or ASIC-like channels may play a prominent role in sour-taste transduction.

Acid-activated cation currents in rat vallate taste receptor cells.

The 5'-ribonucleotide guanosine 5'-monophosphate (GMP) is used widely as an umami taste stimulus and a potent flavor enhancer as it synergistically increases the umami taste elicited by monosodium glutamate. Transduction mechanisms for GMP and its synergy with glutamate are largely unknown. Using whole-cell patch-clamp and Ca(2+) imaging, we examined responses to GMP, glutamate, and a mixture of GMP and glutamate in taste-receptor cells of rat fungiform papillae. Our electrophysiological results showed that GMP induces responses that are similar to those of glutamate, e.g., an outward current, an inward current, or a biphasic response. Our Ca(2+) imaging results showed that applications of GMP, glutamate, and the mixture increased intracellular Ca(2+) levels. Interestingly, both patch-clamp and Ca(2+) imaging showed that some taste cells can respond to GMP and glutamate independently, indicating that glutamate and GMP likely activate different receptors. Simultaneous application of GMP and glutamate resulted in synergistic responses in a subset of cells; both response intensity and number of responding cells were increased. Most responses to GMP, as well as the synergy between GMP and glutamate, were suppressed by 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) in patch-clamp recordings. Together, our results suggest that intracellular cAMP- and Ca(2+)-mediated pathways are involved in umami taste transduction for GMP and its synergistic responses with glutamate.

Responses to di-sodium guanosine 5'-monophosphate and monosodium L-glutamate in taste receptor cells of rat fungiform papillae.

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells.

Extracellular nucleotides such as ATP are the signaling molecules which bind to membrane receptors (P2X ligand-gated ion channels and G-protein-coupled P2Y families). In the gustatory system, it is known that P2X receptors are expressed exclusively in nerve fibers innervating the taste buds. Also, P2Y receptors are suggested to play some important roles in taste signal transductions on the basis of the physiological studies. In the present study, we examined the expression patterns of P2Y1 receptor subtype by using reverse transcript polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR analyses showed that P2Y1 receptor mRNAs appeared in circumvallate papillae. P2Y1 receptor mRNA was detected in a subset of taste bud cells by in situ hybridization. By immunohistochemical analyses, P2Y1 receptor was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. We showed that ATP induced a biphasic intracellular Ca2+ increase in taste cells by a Ca2+ imaging method. Furthermore, we showed by double-immunolabeling methods that P2Y1-expressing cells coexpressed both IP3R3 and SNAP-25. These results suggest that ATP may activate P2Y receptors resulting in Ca2+ release from internal stores via IP3R3. Since many SNAP-25-immunoreactive taste bud cells coexpressed P2Y1 immunoreactivity, it is suggested that P2Y1-expressing cells may possess synapses with afferent nerve fibers. The results of the present study suggest that P2Y1 receptor may play some roles in ATP-mediated signal transductions between taste bud cells and afferent taste fibers.

Expression of P2Y1 receptors in rat taste buds.

Downstream signaling effectors for umami taste.

Acetylcholine and acetylcholine receptors in taste receptor cells.

Acetylcholine (ACh) is well established as a neurotransmitter and/or neuromodulator in various organs. Previously, it has been shown by Ogura (J Neurophysiol 87:2643-2649, 2002) that in both physiological and immunohistochemical studies the muscarinic acetylcholine (ACh) receptor is present in taste receptor cells. However, it has not been determined if ACh is released locally from taste receptor cells and/or surrounding nerve fibers. In this study we investigated the sites of ACh release in mouse taste tissue using the antisera against vesicular ACh transporter (VAChT), a key element of ACh-containing vesicles. Our data show that VAChT-immunoreactivity is present in many taste receptor cells, including cells expressing the transient receptor potential channel M5 (TRPM5). In taste cells, VAChT-immunoreactivity was colocalized with the immunoreactivity to choline-acetyltransferase (ChAT), which synthesizes ACh. Additionally, enhanced green fluorescent protein (eGFP) was detected in the taste cells of BAC-transgenic mice, in which eGFP was placed under the control of endogenous ChAT transcriptional regulatory elements (ChAT(BAC)-eGFP mice). Furthermore, many ChAT-immunolabeled taste cells also reacted to an antibody against the vesicle-associated membrane protein synaptobrevin-2. These data suggest that ACh-containing vesicles are present in taste receptor cells and ACh release from taste cells may play a role in autocrine and/or paracrine cell-to-cell communication. In addition, certain nerve fibers surrounding or within taste buds were immunoreactive for the VAChT antibody. Some of these fibers were also immunolabeled with antibody against calcitonin gene-related peptide (CGRP), a marker for trigeminal peptidergic fibers. Thus, functions of taste receptor cells could be modulated by trigeminal fibers via ACh release as well.

Immuno-localization of vesicular acetylcholine transporter in mouse taste cells and adjacent nerve fibers: indication of acetylcholine release.

Inhaled airborne irritants elicit sensory responses in trigeminal nerves innervating the nasal epithelium, leading to protective reflexes. The sensory mechanisms involved in the detection of odorous irritants are poorly understood. We identified a large population of solitary chemosensory cells expressing the transient receptor potential channel M5 (TRPM5) using transgenic mice where the promoter of TRPM5 drives the expression of green fluorescent protein (GFP). Most of these solitary chemosensory cells lie in the anterior nasal cavity. These GFP-labeled solitary chemosensory cells exhibited immunoreactivity for synaptobrevin-2, a vesicle-associated membrane protein important for synaptic transmission. Concomitantly, we found trigeminal nerve fibers apposed closely to the solitary chemosensory cells, indicating potential transmission of sensory information to trigeminal fibers. In addition, stimulation of the nasal cavity with high concentrations (0.5-5 mM) of a variety of odorants elicited event-related potentials (ERPs) in areas rich in TRPM5-expressing solitary chemosensory cells. Furthermore, odorous chemicals and trigeminal stimuli induced changes in intracellular Ca(2+) levels in isolated TRPM5-expressing solitary chemosensory cells in a concentration-dependent manner. Together, our data show that the TRPM5-expressing cells respond to a variety of chemicals at high exposure levels typical of irritants and are positioned in the nasal cavity appropriately to monitor inhaled air quality.

TRPM5-expressing solitary chemosensory cells respond to odorous irritants.

The sense of taste is crucial in an animal's determination as to what is edible and what is not. This gustatory function is especially important in goldfish, who utilize a sophisticated oropharyngeal sorting mechanism to separate food from substrate material. The computational aspects of this detection are carried out by the medullary vagal lobe, which is a large, laminated structure combining elements of both the gustatory nucleus of the solitary tract and the nucleus ambiguus. The sensory layers of the vagal lobe are coupled to the motor layers via a simple reflex arc. Details of this reflex circuit were investigated with histology and calcium imaging. Biocytin injections into the motor layer labeled vagal reflex interneurons that have radially directed dendrites ramifying within the layers of primary afferent terminals. Axons of reflex interneurons extend radially inward to terminate onto both vagal motoneurons and small, GABAergic interneurons in the motor layer. Functional imaging shows increases in intracellular Ca++ of vagal motoneurons following electrical stimulation in the sensory layer. These responses were suppressed under Ca(++)-free conditions and by interruption of the axons bridging between the sensory and motor layers. Pharmacological experiments showed that glutamate acting via (+/-)-alpha-amino-3-hydroxy- 5-ethylisoxazole-4-propioinc acid (AMPA)/kainate and N-methyl-D-aspartic acid (NMDA) receptors mediate neurotransmission between reflex interneurons and vagal motoneurons. Thus, the vagal gustatory portion of the viscerosensory complex is linked to branchiomotor neurons of the pharynx via a glutamatergic interneuronal system.

Vagal gustatory reflex circuits for intraoral food sorting behavior in the goldfish: cellular organization and neurotransmitters.

Controlling stimulus access to sensory organs allows animals to optimize sensory reception and prevent damage. The vomeronasal organ (VNO) detects pheromones and other semiochemicals to regulate innate social and sexual behaviors. This semiochemical detection generally requires the VNO to draw in chemical fluids, such as bodily secretions, which are complex in composition and can be contaminated. Little is known about whether and how chemical constituents are monitored to regulate the fluid access to the VNO. Using transgenic mice and immunolabeling, we found that solitary chemosensory cells (SCCs) reside densely at the entrance duct of the VNO. In this region, most of the intraepithelial trigeminal fibers innervate the SCCs, indicating that SCCs relay sensory information onto the trigeminal fibers. These SCCs express transient receptor potential channel M5 (TRPM5) and the phospholipase C (PLC) beta2 signaling pathway. Additionally, the SCCs express choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) for synthesizing and packaging acetylcholine, a potential transmitter. In intracellular Ca2+ imaging, the SCCs responded to various chemical stimuli including high concentrations of odorants and bitter compounds. The responses were suppressed significantly by a PLC inhibitor, suggesting involvement of the PLC pathway. Further, we developed a quantitative dye assay to show that the amount of stimulus fluid that entered the VNOs of behaving mice is inversely correlated to the concentration of odorous and bitter substances in the fluid. Genetic knockout and pharmacological inhibition of TRPM5 resulted in larger amounts of bitter compounds entering the VNOs. Our data uncovered that chemoreception of fluid constituents regulates chemical access to the VNO and plays an important role in limiting the access of non-specific irritating and harmful substances. Our results also provide new insight into the emerging role of SCCs in chemoreception and regulation of physiological actions.

Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells.

The mammalian olfactory epithelium is made up of ciliated olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells. Previously, we reported that a population of nonneuronal microvillous cells expresses transient receptor potential channel M5 (TRPM5). Using transgenic mice and immunocytochemical labeling, we identify that these cells are cholinergic, expressing the signature markers of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. This result suggests that acetylcholine (ACh) can be synthesized and released locally to modulate activities of neighboring supporting cells and OSNs. In Ca(2+) imaging experiments, ACh induced increases in intracellular Ca(2+) levels in 78% of isolated supporting cells tested in a concentration-dependent manner. Atropine, a muscarinic ACh receptor (mAChR) antagonist suppressed the ACh responses. In contrast, ACh did not induce or potentiate Ca(2+) increases in OSNs. Instead ACh suppressed the Ca(2+) increases induced by the adenylyl cyclase activator forskolin in some OSNs. Supporting these results, we found differential expression of mAChR subtypes in supporting cells and OSNs using subtype-specific antibodies against M(1) through M(5) mAChRs. Furthermore, we found that various chemicals, bacterial lysate, and cold saline induced Ca(2+) increases in TRPM5/ChAT-expressing microvillous cells. Taken together, our data suggest that TRPM5/ChAT-expressing microvillous cells react to certain chemical or thermal stimuli and release ACh to modulate activities of neighboring supporting cells and OSNs via mAChRs. Our studies reveal an intrinsic and potentially potent mechanism linking external stimulation to cholinergic modulation of activities in the olfactory epithelium.

Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells.

Quantification of nerve fibers in peripheral and central nervous systems is important for the understanding of neuronal function, organization and pathological changes. However, current methods to quantify nerve fibers are resource-intensive and often provide an indirect measurement of nerve fiber density. Here, we describe an automated and efficient method for nerve fiber quantification, which we developed by making use of widely available software and analytical techniques, including Hessian-based feature extraction in NIH ImageJ and line intensity scan analysis. The combined use of these analytical tools through an automated routine enables reliable detection and quantification of nerve fibers from low magnification, non-uniformly labeled epifluorescence images. This allows for time-efficient determination of nerve density and also comparative analysis in large brain structures, such as hippocampus or between various regions of neural circuitry. Using this method, we have obtained accurate measurements of cholinergic fiber density in hippocampus and a large area of cortex in mouse brain sections immunolabeled with an antibody against the vesicular acetylcholine transporter (VAChT). The density values are comparable among animals tested, showing a high degree of reproducibility. Because our method can be performed at relatively low cost and in large tissue sections where nerve fibers can be labeled by various antibodies or visualized by expression of reporter proteins, such as green fluorescent protein in transgenic mice, we expect our method to be broadly useful in both research and clinical investigation. To our knowledge, this is the first method to reliably quantify nerve fibers through a rapid and automated protocol.

Article	Year
Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors. Journal of neurophysiology\| PubMed ID: 12037167	2002
Taste receptor cell responses to the bitter stimulus denatonium involve Ca2+ influx via store-operated channels. Journal of neurophysiology\| PubMed ID: 12037215	2002
Acid-activated cation currents in rat vallate taste receptor cells. Journal of neurophysiology\| PubMed ID: 12091539	2002
Responses to di-sodium guanosine 5'-monophosphate and monosodium L-glutamate in taste receptor cells of rat fungiform papillae. Journal of neurophysiology\| PubMed ID: 12626621	2003
Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells. Cell calcium\| PubMed ID: 12765699	2003
Expression of P2Y1 receptors in rat taste buds. Histochemistry and cell biology\| PubMed ID: 15103469	2004
Downstream signaling effectors for umami taste. Chemical senses\| PubMed ID: 15738174	2005
Acetylcholine and acetylcholine receptors in taste receptor cells. Chemical senses\| PubMed ID: 15738185	2005
Immuno-localization of vesicular acetylcholine transporter in mouse taste cells and adjacent nerve fibers: indication of acetylcholine release. Cell and tissue research\| PubMed ID: 17704949	2007
TRPM5-expressing solitary chemosensory cells respond to odorous irritants. Journal of neurophysiology\| PubMed ID: 18160424	2008
Vagal gustatory reflex circuits for intraoral food sorting behavior in the goldfish: cellular organization and neurotransmitters. The Journal of comparative neurology\| PubMed ID: 19598285	2009
Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells. PloS one\| PubMed ID: 20689832	2010
Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells. Journal of neurophysiology\| PubMed ID: 21676931	2011
Automated measurement of nerve fiber density using line intensity scan analysis. Journal of neuroscience methods\| PubMed ID: 22613744	2012

Tatsuya Ogura

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