Selectively Imaging Cranial Sensory Ganglion Neurons Using AAV-PHP.S.

Because of their ease of use, adeno-associated viruses (AAVs) are indispensable tools for much of neuroscience. Yet AAVs have been used relatively little to study the identities and connectivity of peripheral sensory neurons, principally because methods to selectively target peripheral neurons have been limited. The introduction of the AAV-PHP.S capsid with enhanced tropism for peripheral neurons (Chan et al., 2017) offered a solution, which we further elaborate here. Using AAV-PHP.S with GFP or mScarlet fluorescent proteins, we show that the mouse sensory ganglia for cranial nerves V, VII, IX, and X are targeted. Pseudounipolar neurons of both somatic and visceral origin, but not satellite glia, express the reporters. One week after virus injection, ≈66% of geniculate ganglion neurons were transduced. Fluorescent reporters were transported along the central and peripheral axons of these sensory neurons, permitting visualization of terminals at high resolution, and in intact, cleared brain using light sheet microscopy. Further, using a Cre-dependent reporter, we demonstrate by anatomic and functional criteria, that expression is in a cell type-selective manner. Finally, we integrate earlier neuroanatomical and molecular data with in vivo Ca2+ imaging to demonstrate the sensory characteristics of geniculate ganglion auricular neurons, which were previously undocumented. Our analyses suggest that the AAV-PHP.S serotype will be a powerful tool for anatomically and functionally mapping the receptive fields and circuits of the expanding numbers of molecular subtypes of many somatosensory and viscerosensory neurons that continue to be defined via single-cell RNA sequencing.

"Tripartite Synapses" in Taste Buds: A Role for Type I Glial-like Taste Cells.

In mammalian taste buds, Type I cells comprise half of all cells. These are termed "glial-like" based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. Using Gad2;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ≈28% Type III cells expressing weakly). In dissociated taste buds, GCaMP+ Type I cells responded to bath-applied ATP (10-100 µm) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 µm cycloheximide, 1 mm denatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 µm). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses.SIGNIFICANCE STATEMENT Most studies of taste buds view the chemosensitive excitable cells that express taste receptors as the sole mediators of taste detection and transmission to the CNS. Type I "glial-like" cells, with their ensheathing morphology, are mostly viewed as responsible for clearing neurotransmitters and as the "glue" holding the taste bud together. In the present study, we demonstrate that, when intact taste buds respond to their natural stimuli, Type I cells sense the activation of the chemosensory cells by detecting the afferent transmitter. Because Type I cells synthesize GABA, a known gliotransmitter, and cognate receptors are present on both presynaptic and postsynaptic elements, Type I cells may participate in GABAergic synaptic transmission in the manner of astrocytes at tripartite synapses.

Is there a role for GABA in peripheral taste processing.

In the peripheral neurons and circuits for hearing, balance, touch and pain, GABA plays diverse and important roles. In some cases, GABA is an essential player in the maintenance of sensory receptors and afferent neurons. In other instances, GABA modulates the sensory signal before it reaches CNS neurons. And in yet other instances, tonic GABA-mediated signals set the resting tone and excitability of afferent neurons. GABAA receptors are present on gustatory afferent neurons that carry taste signals from taste buds to central circuits in the brainstem. Yet, the functional significance of these receptors is unexplored. Here, I outline some of the roles of GABA in other peripheral sensory systems. I then consider whether similar functions may be ascribed to GABA signaling in the taste periphery.

NIH Workshop Report: sensory nutrition and disease.

In November 2019, the NIH held the "Sensory Nutrition and Disease" workshop to challenge multidisciplinary researchers working at the interface of sensory science, food science, psychology, neuroscience, nutrition, and health sciences to explore how chemosensation influences dietary choice and health. This report summarizes deliberations of the workshop, as well as follow-up discussion in the wake of the current pandemic. Three topics were addressed: A) the need to optimize human chemosensory testing and assessment, B) the plasticity of chemosensory systems, and C) the interplay of chemosensory signals, cognitive signals, dietary intake, and metabolism. Several ways to advance sensory nutrition research emerged from the workshop: 1) refining methods to measure chemosensation in large cohort studies and validating measures that reflect perception of complex chemosensations relevant to dietary choice; 2) characterizing interindividual differences in chemosensory function and how they affect ingestive behaviors, health, and disease risk; 3) defining circuit-level organization and function that link and interact with gustatory, olfactory, homeostatic, visceral, and cognitive systems; and 4) discovering new ligands for chemosensory receptors (e.g., those produced by the microbiome) and cataloging cell types expressing these receptors. Several of these priorities were made more urgent by the current pandemic because infection with sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease of 2019 has direct short- and perhaps long-term effects on flavor perception. There is increasing evidence of functional interactions between the chemosensory and nutritional sciences. Better characterization of this interface is expected to yield insights to promote health, mitigate disease risk, and guide nutrition policy.

Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells.

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.

The Role of the Anion in Salt (NaCl) Detection by Mouse Taste Buds.

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50-500 mm) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca2+ imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca2+ in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl- rather than Na+ Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl- remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste.SIGNIFICANCE STATEMENT Sodium and chloride are essential nutrients and must be regularly consumed to replace excreted NaCl. Thus, understanding salt taste, which informs salt appetite, is important from a fundamental sensory perspective and forms the basis for interventions to replace/reduce excess Na+ consumption. This study examines responses to NaCl in a semi-intact preparation of mouse taste buds. We identify taste cells that respond to NaCl in the presence of amiloride, which is significant because much of human salt taste also is amiloride-insensitive. Further, we demonstrate that Cl-, not Na+, generates these amiloride-insensitive salt taste responses. Intriguingly, choline chloride, a commercial salt taste enhancer, is also a highly effective stimulus for these cells.

Cell-Based Therapy Restores Olfactory Function in an Inducible Model of Hyposmia.

Stem cell-based therapies have been proposed as a strategy to replace damaged tissues, especially in the nervous system. A primary sensory modality, olfaction, is impaired in 12% of the US population, but lacks treatment options. We report here the development of a novel mouse model of inducible hyposmia and demonstrate that purified tissue-specific stem cells delivered intranasally engraft to produce olfactory neurons, achieving recovery of function. Adult mice were rendered hyposmic by conditional deletion of the ciliopathy-related IFT88 gene in the olfactory sensory neuron lineage and following experimentally induced olfactory injury, received either vehicle or stem cell infusion intranasally. Engraftment-derived olfactory neurons were identified histologically, and functional improvements were measured via electrophysiology and behavioral assay. We further explored mechanisms in culture that promote expansion of engraftment-competent adult olfactory basal progenitor cells. These findings provide a basis for translational research on propagating adult tissue-specific sensory progenitor cells and testing their therapeutic potential.

Recognizing Taste: Coding Patterns Along the Neural Axis in Mammals.

The gustatory system encodes information about chemical identity, nutritional value, and concentration of sensory stimuli before transmitting the signal from taste buds to central neurons that process and transform the signal. Deciphering the coding logic for taste quality requires examining responses at each level along the neural axis-from peripheral sensory organs to gustatory cortex. From the earliest single-fiber recordings, it was clear that some afferent neurons respond uniquely and others to stimuli of multiple qualities. There is frequently a "best stimulus" for a given neuron, leading to the suggestion that taste exhibits "labeled line coding." In the extreme, a strict "labeled line" requires neurons and pathways dedicated to single qualities (e.g., sweet, bitter, etc.). At the other end of the spectrum, "across-fiber," "combinatorial," or "ensemble" coding requires minimal specific information to be imparted by a single neuron. Instead, taste quality information is encoded by simultaneous activity in ensembles of afferent fibers. Further, "temporal coding" models have proposed that certain features of taste quality may be embedded in the cadence of impulse activity. Taste receptor proteins are often expressed in nonoverlapping sets of cells in taste buds apparently supporting "labeled lines." Yet, taste buds include both narrowly and broadly tuned cells. As gustatory signals proceed to the hindbrain and on to higher centers, coding becomes more distributed and temporal patterns of activity become important. Here, we present the conundrum of taste coding in the light of current electrophysiological and imaging techniques at several levels of the gustatory processing pathway.

Oral thermosensing by murine trigeminal neurons: modulation by capsaicin, menthol and mustard oil.

KEY POINTS: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range. Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices. There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds. Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. ABSTRACT: When consumed with foods, mint, mustard, and chili peppers generate pronounced oral thermosensations. Here we recorded responses in mouse trigeminal ganglion neurons to investigate interactions between thermal sensing and the active ingredients of these plants - menthol, allyl isothiocyanate (AITC), and capsaicin, respectively - at concentrations found in foods and commercial hygiene products. We carried out in vivo confocal calcium imaging of trigeminal ganglia in which neurons express GCaMP3 or GCAMP6s and recorded their responses to oral stimulation with thermal and the above chemesthetic stimuli. In the V3 (oral sensory) region of the ganglion, thermoreceptive neurons accounted for ∼10% of imaged neurons. We categorized them into three distinct classes: cool-responsive and warm-responsive thermosensors, and nociceptors (responsive only to temperatures ≥43-45 °C). Menthol, AITC, and capsaicin also elicited robust calcium responses that differed markedly in their latencies and durations. Most of the neurons that responded to these chemesthetic stimuli were also thermosensitive. Capsaicin and AITC increased the numbers of warm-responding neurons and shifted the nociceptor threshold to lower temperatures. Menthol attenuated the responses in all classes of thermoreceptors. Our data show that while individual neurons may respond to a narrow temperature range (or even bimodally), taken collectively, the population is able to report on graded changes of temperature. Our findings also substantiate an explanation for the thermal sensations experienced when one consumes pungent spices or mint.

Transcriptomes and neurotransmitter profiles of classes of gustatory and somatosensory neurons in the geniculate ganglion.

Taste buds are innervated by neurons whose cell bodies reside in cranial sensory ganglia. Studies on the functional properties and connectivity of these neurons are hindered by the lack of markers to define their molecular identities and classes. The mouse geniculate ganglion contains chemosensory neurons innervating lingual and palatal taste buds and somatosensory neurons innervating the pinna. Here, we report single cell RNA sequencing of geniculate ganglion neurons. Using unbiased transcriptome analyses, we show a pronounced separation between two major clusters which, by anterograde labeling, correspond to gustatory and somatosensory neurons. Among the gustatory neurons, three subclusters are present, each with its own complement of transcription factors and neurotransmitter response profiles. The smallest subcluster expresses both gustatory- and mechanosensory-related genes, suggesting a novel type of sensory neuron. We identify several markers to help dissect the functional distinctions among gustatory neurons and address questions regarding target interactions and taste coding.Characterization of gustatory neural pathways has suffered due to a lack of molecular markers. Here, the authors report single cell RNA sequencing and unbiased transcriptome analyses to reveal major distinctions between gustatory and somatosensory neurons and subclusters of gustatory neurons with unique molecular and functional profiles.

Taste buds: cells, signals and synapses.

The past decade has witnessed a consolidation and refinement of the extraordinary progress made in taste research. This Review describes recent advances in our understanding of taste receptors, taste buds, and the connections between taste buds and sensory afferent fibres. The article discusses new findings regarding the cellular mechanisms for detecting tastes, new data on the transmitters involved in taste processing and new studies that address longstanding arguments about taste coding.

Contribution of Polycomb group proteins to olfactory basal stem cell self-renewal in a novel c-KIT+ culture model and in vivo.

Olfactory epithelium (OE) has a lifelong capacity for neurogenesis due to the presence of basal stem cells. Despite the ability to generate short-term cultures, the successful in vitro expansion of purified stem cells from adult OE has not been reported. We sought to establish expansion-competent OE stem cell cultures to facilitate further study of the mechanisms and cell populations important in OE renewal. Successful cultures were prepared using adult mouse basal cells selected for expression of c-KIT. We show that c-KIT signaling regulates self-renewal capacity and prevents neurodifferentiation in culture. Inhibition of TGFß family signaling, a known negative regulator of embryonic basal cells, is also necessary for maintenance of the proliferative, undifferentiated state in vitro Characterizing successful cultures, we identified expression of BMI1 and other Polycomb proteins not previously identified in olfactory basal cells but known to be essential for self-renewal in other stem cell populations. Inducible fate mapping demonstrates that BMI1 is expressed in vivo by multipotent OE progenitors, validating our culture model. These findings provide mechanistic insights into the renewal and potency of olfactory stem cells.

Differentiation potential of individual olfactory c-Kit+ progenitors determined via multicolor lineage tracing.

Olfactory tissue undergoes lifelong renewal, due to the presence of basal neural stem cells. Multiple categories of globose basal stem cells have been identified, expressing markers such as Lgr5, Ascl1, GBC-2, and c-Kit. The differentiation potential of individual globose cells has remained unclear. Here, we utilized Cre/loxP lineage tracing with a multicolor reporter system to define c-Kit+ cell contributions at clonal resolution. We determined that reporter expression permitted identification of c-Kit derived progeny with fine cellular detail, and that clones were found to be comprised by neurons only, microvillar cells only, microvillar cells and neurons, or gland/duct cells. Quantification of reporter-labeled cells indicated that c-Kit+ cells behave as transit amplifying or immediate precursors, although we also found evidence for longer-term c-Kit+ cell contributions. Our results from the application of multicolor fate mapping delineate the clonal contributions of c-Kit+ cells to olfactory epithelial renewal, and provide novel insight into tissue maintenance of an adult neuroepithelium.

Breadth of tuning in taste afferent neurons varies with stimulus strength.

Gustatory stimuli are detected by taste buds and transmitted to the hindbrain via sensory afferent neurons. Whether each taste quality (sweet, bitter and so on) is encoded by separate neurons ('labelled lines') remains controversial. We used mice expressing GCaMP3 in geniculate ganglion sensory neurons to investigate taste-evoked activity. Using confocal calcium imaging, we recorded responses to oral stimulation with prototypic taste stimuli. Up to 69% of neurons respond to multiple tastants. Moreover, neurons tuned to a single taste quality at low concentration become more broadly tuned when stimuli are presented at higher concentration. Responses to sucrose and monosodium glutamate are most related. Although mice prefer dilute NaCl solutions and avoid concentrated NaCl, we found no evidence for two separate populations of sensory neurons that encode this distinction. Altogether, our data suggest that taste is encoded by activity in patterns of peripheral sensory neurons and challenge the notion of strict labelled line coding.

Cocaine decreases saccharin preference without altering sweet taste sensitivity.

In rodents, saccharin consumption is suppressed when the sweet taste stimulus is paired with moderate doses of cocaine. Several hypotheses have been used to explain the seemingly contradictory effect of decreased consumption of a normally preferred substance following a highly rewarding drug. A common theme across these hypotheses is that saccharin is interpreted as less rewarding after cocaine pairing. We considered the alternative possibility that suppression is caused not by a change in reward circuitry, but rather by a change in taste detection, for instance by altering the afferent taste response and decreasing sensitivity to sweet taste stimuli. To evaluate this possibility, we measured saccharin taste sensitivity of mice before and after a standard cocaine-pairing paradigm. We measured taste sensitivity using a brief-access lickometer equipped with multiple concentrations of saccharin solution and established concentration-response curves before and after saccharin-cocaine pairing. Our results indicate that the EC50 for saccharin was unaltered following pairing. Instead, the avidity of licking saccharin, an indicator of motivation, was depressed. Latency to first-lick, a negative indicator of motivation, was also dramatically increased. Thus, our findings are consistent with the interpretation that saccharin-cocaine pairing results in devaluing of the sweet taste reward.

Oxytocin decreases sweet taste sensitivity in mice.

Oxytocin (OXT) suppresses food intake and lack of OXT leads to overconsumption of sucrose. Taste bud cells were recently discovered to express OXT-receptor. In the present study we tested whether administering OXT to wild-type mice affects their licking behavior for tastants in a paradigm designed to be sensitive to taste perception. We injected C57BL/6J mice intraperitoneally (i.p.) with 10mg/kg OXT and assayed their brief-access lick responses, motivated by water deprivation, to NaCl (300mM), citric acid (20mM), quinine (0.3mM), saccharin (10mM), and a mix of MSG and IMP (100mM and 0.5mM respectively). OXT had no effect on licking for NaCl, citric acid, or quinine. A possible effect of OXT on saccharin and MSG+IMP was difficult to interpret due to unexpectedly low lick rates to water (the vehicle for all taste solutions), likely caused by the use of a high OXT dose that suppressed licking and other behaviors. A subsequent experiment focused on another preferred tastant, sucrose, and employed a much lower OXT dose (0.1mg/kg). This modification, based on our measurements of plasma OXT following i.p. injection, permitted us to elevate plasma [OXT] sufficiently to preferentially activate taste bud cells. OXT at this low dose significantly reduced licking responses to 0.3M sucrose, and overall shifted the sucrose concentration - behavioral response curves rightward (mean EC50saline=0.362M vs. EC50OXT=0.466M). Males did not differ from females under any condition in this study. We propose that circulating oxytocin is another factor that modulates taste-based behavior.

A permeability barrier surrounds taste buds in lingual epithelia.

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste.

Synaptic communication and signal processing among sensory cells in taste buds.

Taste buds (sensory structures embedded in oral epithelium) show a remarkable diversity of transmitters synthesized and secreted locally. The known transmitters accumulate in a cell type selective manner, with 5-HT and noradrenaline being limited to presynaptic cells, GABA being synthesized in both presynaptic and glial-like cells, and acetylcholine and ATP used for signalling by receptor cells. Each of these transmitters participates in local negative or positive feedback circuits that target particular cell types. Overall, the role of ATP is the best elucidated. ATP serves as a principal afferent transmitter, and also is the key trigger for autocrine positive feedback and paracrine circuits that result in potentiation (via adenosine) or inhibition (via GABA or 5-HT). While many of the cellular receptors and mechanisms for these circuits are known, their impact on sensory detection and perception remains to be elaborated in most instances. This brief review examines what is known, and some of the open questions and controversies surrounding the transmitters and circuits of the taste periphery.