Optogenetic Stimulation of Type I GAD65+ Cells in Taste Buds Activates Gustatory Neurons and Drives Appetitive Licking Behavior in Sodium-Depleted Mice.

Mammalian taste buds are comprised of specialized neuroepithelial cells that act as sensors for molecules that provide nutrition (e.g., carbohydrates, amino acids, and salts) and those that are potentially harmful (e.g., certain plant compounds and strong acids). Type II and III taste bud cells (TBCs) detect molecules described by humans as "sweet," "bitter," "umami," and "sour." TBCs that detect metallic ions, described by humans as "salty," are undefined. Historically, type I glial-like TBCs have been thought to play a supportive role in the taste bud, but little research has been done to explore their role in taste transduction. Some evidence implies that type I cells may detect sodium (Na+) via an amiloride-sensitive mechanism, suggesting they play a role in Na+ taste transduction. We used an optogenetic approach to study type I TBCs by driving the expression of the light-sensitive channelrhodopsin-2 (ChR2) in type I GAD65+ TBCs of male and female mice. Optogenetic stimulation of GAD65+ TBCs increased chorda tympani nerve activity and activated gustatory neurons in the rostral nucleus tractus solitarius. "N neurons," whose NaCl responses were blocked by the amiloride analog benzamil, responded robustly to light stimulation of GAD65+ TBCs on the anterior tongue. Two-bottle preference tests were conducted under Na+-replete and Na+-deplete conditions to assess the behavioral impact of optogenetic stimulation of GAD65+ TBCs. Under Na+-deplete conditions GAD65-ChR2-EYFP mice displayed a robust preference for H2O illuminated with 470 nm light versus nonilluminated H2O, suggesting that type I glial-like TBCs are sufficient for driving a behavior that resembles Na+ appetite.SIGNIFICANCE STATEMENT This is the first investigation on the role of type I GAD65+ taste bud cells (TBCs) in taste-mediated physiology and behavior via optogenetics. It details the first definitive evidence that selective optogenetic stimulation of glial-like GAD65+ TBCs evokes neural activity and modulates behavior. Optogenetic stimulation of GAD65+ TBCs on the anterior tongue had the strongest effect on gustatory neurons that responded best to NaCl stimulation through a benzamil-sensitive mechanism. Na+-depleted mice showed robust preferences to "light taste" (H2O illuminated with 470 nm light vs nonilluminated H2O), suggesting that the activation of GAD65+ cells may generate a salt-taste sensation in the brain. Together, our results shed new light on the role of GAD65+ TBCs in gustatory transduction and taste-mediated behavior.

Movement assay for the undergraduate neuroscience laboratory.

Described is a design for easy-to-construct apparatus that measures movement of flying insects suitable for the undergraduate teaching laboratory. The system does not require the purchase of specialized scientific equipment or software. The apparatus can be constructed and operated without advanced knowledge in electronics or programming. The goal of this apparatus was to expand upon previous research detecting insect flight in response to radiation. We improved upon the quantification and resolution of flight across differing intensities of white light. All of this was achieved using low-cost and commonly available materials and open-source software to collect and analyze data. The only substantial prerequisites for this design are a PC with a 3.5 mm microphone input and an understanding of basic electrical connections. The apparatus was validated with comparative physiological data from two different species of butterfly.

An open-source lickometer and microstructure analysis program.

Described herein is a design for a user-constructed electronic lickometer, intended to allow users to conduct relatively simple behavioral experiments with rodents while avoiding several common stumbling blocks. Primarily, this system does not require the purchase of specialized scientific equipment or software. Additionally, it is possible for users to construct and operate this lickometer without the prerequisite of advanced knowledge of electronics or programming. Overall, the goal of this apparatus is to provide a simple and affordable alternative for users seeking to study ingestion behaviors in rodents, while still allowing the user to obtain high-resolution data and conduct sophisticated microstructural analysis of the behavior in question. All of this is achieved using low-cost and commonly available materials for the construction of the apparatus itself, and open-source software to collect and analyze data. The only substantial prerequisites for this design are a PC with a 3.5 mm microphone input and a comfortable understanding of power tools. Finally, a validation of the operation of the describe apparatus is included.

C. elegans germ cells divide and differentiate in a folded tissue.

Knowing how stem cells and their progeny are positioned within their tissues is essential for understanding their regulation. One paradigm for stem cell regulation is the C. elegans germline, which is maintained by a pool of germline stem cells in the distal gonad, in a region known as the 'progenitor zone'. The C. elegans germline is widely used as a stem cell model, but the cellular architecture of the progenitor zone has been unclear. Here we characterize this architecture by creating virtual 3D models of the progenitor zone in both sexes. We show that the progenitor zone in adult hermaphrodites is organized like a folded epithelium. The progenitor zone in males is not folded. Analysis of germ cell division shows that daughter cells are born side-by-side along the epithelial-like surface of the germline tissue. Analysis of a key regulator driving differentiation, GLD-1, shows that germ cells in hermaphrodites differentiate along a folded path, with previously described "steps" in GLD-1 expression corresponding to germline folds. Our study provides a three-dimensional view of how C. elegans germ cells progress from stem cell to overt differentiation, with critical implications for regulators driving this transition.

Aging Decreases Chorda-Tympani Nerve Responses to NaCl and Alters Morphology of Fungiform Taste Pores in Rats.

Sensory processing is susceptible to decline with age. The sense of taste is, however, generally thought to be resistant to aging. We investigated how chorda-tympani nerve responses and fungiform-taste pores are affected by aging in the Sprague-Dawley rat, a model system for salt taste. First, we measured chorda-tympani nerve responses to NH4Cl and NaCl solutions in young (3-5 months old) and aged (14-15 months old) rats. Aged rats had significantly attenuated chorda-tympani responses to 0.01, 0.03, 0.1, and 0.3 M NaCl, whereas responses to NH4Cl were statistically similar between age groups. Second, we investigated if fungiform papillae, which harbor taste buds innervated by the chorda-tympani nerve, were affected by aging in "young" (4-7 months old) and "aged" ("aged1" 18 months old and "aged2" 24-28 months old) rats. Using scanning electron microscopy, we found that aging significantly reduced morphological characteristics associated with intact fungiform-taste pores (hillock, rim, pore presence, and open pore). We conclude that the structure and function of the peripheral-taste system may not be as resistant to aging as previously reported.

Thirst Increases Chorda Tympani Responses to Sodium Chloride.

In nature, water is present as a low-salt solution, thus we hypothesized that thirst would increase taste responses to low-salt solutions. We investigated the effect of thirst on the 2 different salt detection mechanisms present in the rat chorda tympani (CT) nerve. The first mechanism is dependent upon the epithelial sodium channel (ENaC), is blocked by benzamil, and is specific to the cation sodium. The second mechanism, while undefined, is independent of ENaC, and detects multiple cations. We expected thirst to increase benzamil-sensitive sodium responses due to mechanistically increasing the benzamil-sensitive ENaC. We recorded CT whole-nerve electrophysiological responses to lingual application of NaCl, KCl (30, 75, 150, 300, 500, and 600 mM), and imitation rainwater in both control and 24-h water-restricted male rats. NaCl solutions were presented in artificial saliva before and after lingual application of 5µM benzamil. Water restriction significantly increased the integrated CT responses to NaCl but not to KCl or imitation rainwater. Consistent with our hypothesis, only the benzamil-sensitive, and not the benzamil-insensitive, CT sodium response significantly increased. Additionally, CT responses to salt were recorded following induction of either osmotic or volemic thirst. Both thirsts significantly enhanced the integrated CT responses to NaCl and KCl, but not imitation rainwater. Interestingly, osmotic and volemic thirsts increased CT responses by increasing both the benzamil-sensitive and benzamil-insensitive CT sodium responses. We propose that thirst increases the sensitivity of the CT nerve to sodium.

Statistical analysis and decoding of neural activity in the rodent geniculate ganglion using a metric-based inference system.

We analyzed the spike discharge patterns of two types of neurons in the rodent peripheral gustatory system, Na specialists (NS) and acid generalists (AG) to lingual stimulation with NaCl, acetic acid, and mixtures of the two stimuli. Previous computational investigations found that both spike rate and spike timing contribute to taste quality coding. These studies used commonly accepted computational methods, but they do not provide a consistent statistical evaluation of spike trains. In this paper, we adopted a new computational framework that treated each spike train as an individual data point for computing summary statistics such as mean and variance in the spike train space. We found that these statistical summaries properly characterized the firing patterns (e. g. template and variability) and quantified the differences between NS and AG neurons. The same framework was also used to assess the discrimination performance of NS and AG neurons and to remove spontaneous background activity or "noise" from the spike train responses. The results indicated that the new metric system provided the desired decoding performance and noise-removal improved stimulus classification accuracy, especially of neurons with high spontaneous rates. In summary, this new method naturally conducts statistical analysis and neural decoding under one consistent framework, and the results demonstrated that individual peripheral-gustatory neurons generate a unique and reliable firing pattern during sensory stimulation and that this pattern can be reliably decoded.

Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ.

BACKGROUND: The signal transduction cascade operational in the vomeronasal organ (VNO) of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2), which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO. RESULTS: Homer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1) and receptor expression enhancing protein 1 (REEP1) were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay. CONCLUSIONS: TRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

Human pheromone detection by the vomeronasal organ: unnecessary for mate selection?

Recently, Foltan and Sedy proposed a hypothesis stating that the adult human VNO is integral to the prevention of inappropriate mate selection. In this commentary, we address the authors' assumption that humans have a functional VNO, that pheromones are detected exclusively by the VNO, and that human pheromones are responsible for negative stimuli during mate selection. After examining the published literature on human vomeronasal function, we argue that their hypothesis is critically flawed. We offer a brief review of the adult human VNO in support of our argument.

Awake intranasal insulin delivery modifies protein complexes and alters memory, anxiety, and olfactory behaviors.

The role of insulin pathways in olfaction is of significant interest with the widespread pathology of diabetes mellitus and its associated metabolic and neuronal comorbidities. The insulin receptor (IR) kinase is expressed at high levels in the olfactory bulb, in which it suppresses a dominant Shaker ion channel (Kv1.3) via tyrosine phosphorylation of critical N- and C-terminal residues. We optimized a 7 d intranasal insulin delivery (IND) in awake mice to ascertain the biochemical and behavioral effects of insulin to this brain region, given that nasal sprays for insulin have been marketed notwithstanding our knowledge of the role of Kv1.3 in olfaction, metabolism, and axon targeting. IND evoked robust phosphorylation of Kv1.3, as well as increased channel protein-protein interactions with IR and postsynaptic density 95. IND-treated mice had an increased short- and long-term object memory recognition, increased anxiolytic behavior, and an increased odor discrimination using an odor habituation protocol but only moderate change in odor threshold using a two-choice paradigm. Unlike Kv1.3 gene-targeted deletion that alters metabolism, adiposity, and axonal targeting to defined olfactory glomeruli, suppression of Kv1.3 via IND had no effect on body weight nor the size and number of M72 glomeruli or the route of its sensory axon projections. There was no evidence of altered expression of sensory neurons in the epithelium. In mice made prediabetic via diet-induced obesity, IND was no longer effective in increasing long-term object memory recognition nor increasing anxiolytic behavior, suggesting state dependency or a degree of insulin resistance related to these behaviors.

The effects of analgesic supplements on neural activity in the main olfactory bulb of the mouse.

We evaluated ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), as an antinociceptive supplement to chloral hydrate anesthesia in mouse. Effects of ketoprofen on main olfactory bulb (MOB) neuronal spontaneous activity were investigated using extracellular recordings in mouse in vivo. These effects were compared with those of another nociceptive supplement, the mu-opioid agonist buprenorphine. Ketoprofen (100 or 200 mg/kg) did not significantly alter MOB single-unit spontaneous rates in either ICR or C57BL/6J mice. In contrast, buprenorphine, at doses of 0.02, 0.05, and 0.20 mg/kg, inhibited MOB neuronal spontaneous rates by 19%, 49%, and 57%, respectively. Neither drug altered the temporal patterning of single-unit spike trains, as measured by the interspike interval (ISI) coefficient of variation (CV). We also investigated the ability of ketoprofen and buprenorphine to induce antinociception in the anesthetized mouse. The electroencephalogram (EEG) was used to measure the anesthetic plane. Both ketoprofen and buprenorphine altered the EEG trace and ketoprofen altered the power spectrum in a manner consistent with deepening anesthesia. Lastly, when applied at the time of anesthesia induction, ketoprofen decreased the amount of chloral hydrate necessary to maintain a defined anesthetic plane during the rest of the experiment. These results suggest that ketoprofen induces antinociception under chloral hydrate anesthesia without significantly inhibiting spontaneous activity of MOB neurons. Ketoprofen is therefore suitable as an antinociceptive supplement to chloral hydrate anesthesia during in vivo electrophysiologic recordings of the mouse MOB.

In vivo preparation and identification of mitral cells in the main olfactory bulb of the mouse.

The mouse main olfactory bulb (MOB) is commonly used as a mammalian model to study olfactory processing. The genetic techniques available with the mouse make its MOB a powerful model for analysis of neuronal circuitry. The mouse has been used as a mammalian model for all types of MOB neurons, but especially to study the activity of mitral cells. However, mouse mitral cell activity is most commonly studied in vitro. Therefore, we aimed to develop a protocol to record the activity of antidromically identified mitral cells in mouse in vivo. Currently, such a protocol does not exist. Using extracellular techniques, we report a protocol that is able to record neurons from all mouse MOB layers. Specifically, mitral cell single-units were identified by antidromic activation from the posterior piriform cortex, and their spontaneous activity was recorded for more than 30 min. This protocol is stable enough to record from single-units while buprenorphine was applied both topically to the surface of the MOB and injected systemically.