The behavioral sensitivity of mice to acyclic, monocyclic, and bicyclic monoterpenes.

Monoterpenes are a large class of naturally occurring fragrant molecules. These chemicals are commonly used in olfactory studies to survey neural activity and probe the behavioral limits of odor discrimination. Monoterpenes (typically in the form of essential oils) have been used for centuries for therapeutic purposes and have pivotal roles in various biological and medical applications. Despite their importance for multiple lines of research using rodent models and the role of the olfactory system in detecting these volatile chemicals, the murine sensitivity to monoterpenes remains mostly unexplored. We assayed the ability of C57BL/6J mice to detect nine different monoterpenes (the acyclic monoterpenes: geraniol, citral, and linalool; the monocyclic monoterpenes: r-limonene, s-limonene, and γ-terpinene; and the bicyclic monoterpenes: eucalyptol, α-pinene, and ß-pinene) using a head-fixed Go / No-Go operant conditioning assay. We found that mice can reliably detect monoterpene concentrations in the low parts per billion (ppb) range. Specifically, mice were most sensitive to geraniol (threshold: 0.7 ppb) and least sensitive to γ-terpinene (threshold: 18.1 ppb). These estimations of sensitivity serve to set the lower limit of relevant monoterpene concentrations for functional experiments in mice. To define an upper limit, we estimated the maximum concentrations that a mouse may experience in nature by collating published headspace analyses of monoterpene concentrations emitted from natural sources. We found that natural monoterpenes concentrations typically ranged from ~1 to 1000 ppb. It is our hope that this dataset will help researchers use appropriate monoterpene concentrations for functional studies and provide context for the vapor-phase delivery of these chemicals in studies investigating their biological activity in mice.

Protocol for quantifying the odor detection threshold of mice.

Perceptual measures of odor threshold provide a mechanism to compare sensitivity across species and to gauge stimulus concentrations for functional experiments. Here, we present a protocol to precisely quantify the odor detection threshold of mice. We describe the construction of a head-fixed operant conditioning behavioral rig and provide details of the training and testing procedures. This approach can be used to compare the sensitivity of mice across odorants and to quantify detection differences associated with genetic mutations or pharmacological manipulations. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2023),1 Jennings et al. (2022),2 Williams and Dewan (2020),3 and Dewan et al. (2018).4.

Olfaction: Allosteric modulation.

New research indicates that the odor-evoked responses of human olfactory receptors can be enhanced via the non-competitive binding of an allosteric modulator. This modulatory mechanism adds an additional layer of complexity to the peripheral encoding of odors.

Vasopressin receptor 1a, oxytocin receptor, and oxytocin knockout male and female mice display normal perceptual abilities towards non-social odorants.

Genetic knockouts of the vasopressin receptor 1a (Avpr1a), oxytocin receptor (Oxtr), or oxytocin (Oxt) gene in mice have helped cement the causal relationship between these neuropeptide systems and various social behaviors (e.g., social investigation, recognition, and communication, as well as territoriality and aggression). In mice, these social behaviors depend upon the olfactory system. Thus, it is critical to assess the olfactory capabilities of these knockout models to accurately interpret the observed differences in social behavior. Prior studies utilizing these transgenic mice have sought to test for baseline deficits in olfactory processing; predominantly through use of odor habituation/dishabituation tasks, buried food tests, or investigation assays using non-social odorants. While informative, these assays rely on the animal's intrinsic motivation and locomotor behavior to measure olfactory capabilities and thus, often yield mixed results. Instead, psychophysical analyses using operant conditioning procedures and flow-dilution olfactometry are ideally suited to precisely quantify olfactory perception. In the present study, we used these methods to assess the main olfactory capabilities of adult male and female Avpr1a, Oxtr, and Oxt transgenic mice to volatile non-social odorants. Our results indicate that homozygous and heterozygous knockout mice of all three strains have the same sensitivity and discrimination ability as their wild-type littermates. These data strongly support the hypothesis that the observed social deficits of these global knockout mice are not due to baseline deficits of their main olfactory system.

Estimating the relationship between liquid- and vapor-phase odorant concentrations using a photoionization detector (PID)-based approach.

Olfactory studies frequently utilize odor stimuli consisting of volatiles created from liquid dilutions of various chemicals. A problem arises if the researcher relies on these liquid dilutions to extrapolate vapor concentrations based on ideal gas behavior. For most chemicals, the relationship between liquid and vapor concentration deviates from these laws of proportionality due to interactions between the chemical and the solvent. Here, we describe a method to estimate vapor-phase concentrations of diluted odorants using a photoionization detector. To demonstrate the utility of this method, we assessed the relationship between liquid-/vapor-phase concentrations for 14 odorants (7 alcohols, 1 ester, and 6 aldehydes) in 5 different solvents (water, mineral oil, diethyl phthalate, dipropylene glycol, and propylene glycol). An analysis of 7 additional esters is also included to assess how carbon chain length and functional group, interacts with these solvents (for a total of 105 odorant/solvent pairs). Our resulting equilibrium equations successfully corrected for behavioral sensitivity differences observed in mice tested with the same odorant in different solvents and were overall similar to published measurements using a gas chromatography-based approach. In summary, this method should allow researchers to determine the vapor-phase concentration of diluted odorants and will hopefully assist in more accurate comparisons of odorant concentrations across olfactory studies.

The behavioral sensitivity of mice to acetate esters.

Measures of behavioral sensitivity provide an important guide for choosing the stimulus concentrations used in functional experiments. This information is particularly valuable in the olfactory system as the neural representation of an odorant changes with concentration. This study focuses on acetate esters because they are commonly used to survey neural activity in a variety of olfactory regions, probe the behavioral limits of odor discrimination, and assess odor structure-activity relationships in mice. Despite their frequent use, the relative sensitivity of these odorants in mice is not available. Thus, we assayed the ability of C57BL/6J mice to detect seven different acetates (propyl acetate, butyl acetate, pentyl acetate, hexyl acetate, octyl acetate, isobutyl acetate, and isoamyl acetate) using a head-fixed Go/No-Go operant conditioning assay combined with highly reproducible stimulus delivery. To aid in the accessibility and applicability of our data, we have estimated the vapor-phase concentrations of these odorants in five different solvents using a photoionization detector-based approach. The resulting liquid-/vapor-phase equilibrium equations successfully corrected for behavioral sensitivity differences observed in animals tested with the same odorant in different solvents. We found that mice are most sensitive to isobutyl acetate and least sensitive to propyl acetate. These updated measures of sensitivity will hopefully guide experimenters in choosing appropriate stimulus concentrations for experiments using these odorants.

Sensitive and robust chemical detection using an olfactory brain-computer interface.

When it comes to detecting volatile chemicals, biological olfactory systems far outperform all artificial chemical detection devices in their versatility, speed, and specificity. Consequently, the use of trained animals for chemical detection in security, defense, healthcare, agriculture, and other applications has grown astronomically. However, the use of animals in this capacity requires extensive training and behavior-based communication. Here we propose an alternative strategy, a bio-electronic nose, that capitalizes on the superior capability of the mammalian olfactory system, but bypasses behavioral output by reading olfactory information directly from the brain. We engineered a brain-computer interface that captures neuronal signals from an early stage of olfactory processing in awake mice combined with machine learning techniques to form a sensitive and selective chemical detector. We chronically implanted a grid electrode array on the surface of the mouse olfactory bulb and systematically recorded responses to a large battery of odorants and odorant mixtures across a wide range of concentrations. The bio-electronic nose has a comparable sensitivity to the trained animal and can detect odors on a variable background. We also introduce a novel genetic engineering approach that modifies the relative abundance of particular olfactory receptors in order to improve the sensitivity of our bio-electronic nose for specific chemical targets. Our recordings were stable over months, providing evidence for robust and stable decoding over time. The system also works in freely moving animals, allowing chemical detection to occur in real-world environments. Our bio-electronic nose outperforms current methods in terms of its stability, specificity, and versatility, setting a new standard for chemical detection.

Olfactory signaling via trace amine-associated receptors.

Trace amine-associated receptors (TAARs) are a family of G protein-coupled receptors that function as odorant receptors in the main olfactory system of vertebrates. TAARs are monoallelically expressed in primary sensory neurons where they couple to the same transduction cascade as canonical olfactory receptors and are mapped onto glomeruli within a specific region of the olfactory bulb. TAARs have a high affinity for volatile amines, a class of chemicals that are generated during the decomposition of proteins and are ubiquitous physiological metabolites that are found in body fluids. Thus, amines are proposed to play an important role in intra- and interspecific communication such as signaling the sex of the conspecific, the quality of the food source, or even the proximity of a predator. TAARs have a crucial role in the perception of these behaviorally relevant compounds as the genetic deletion of all or even individual olfactory TAARs can alter the behavioral response and reduce the sensitivity to amines. The small size of this receptor family combined with the ethological relevance of their ligands makes the TAARs an attractive model system for probing olfactory perception. This review will summarize the current knowledge on the olfactory TAARs and discuss whether they represent a unique subsystem within the main olfactory system.

Olfactory Detection Thresholds for Primary Aliphatic Alcohols in Mice.

Probing the neural mechanisms that underlie each sensory system requires the presentation of perceptually appropriate stimulus concentrations. This is particularly relevant in the olfactory system as additional odorant receptors typically respond with increasing stimulus concentrations. Thus, perceptual measures of olfactory sensitivity provide an important guide for functional experiments. This study focuses on aliphatic alcohols because they are commonly used to survey neural activity in a variety of olfactory regions, probe the behavioral limits of odor discrimination, and assess odor-structure activity relationships in mice. However, despite their frequent use, a systematic study of the relative sensitivity of these odorants in mice is not available. Thus, we assayed the ability of C57BL/6J mice to detect a homologous series of primary aliphatic alcohols (1-propanol to 1-heptanol) using a head-fixed Go/No-Go operant conditioning assay combined with highly reproducible stimulus delivery. To aid in the accessibility of our data, we report the animal's threshold to each odorant according to the 1) ideal gas condition, 2) nonideal gas condition (factoring in the activity of the odorant in the solvent), and 3) the liquid dilution of the odorant in the olfactometer. Of the odorants tested, mice were most sensitive to 1-hexanol and least sensitive to 1-butanol. These updated measures of murine sensitivity will hopefully guide experimenters in choosing appropriate stimulus concentrations for experiments using these odorants.

Genetic Depletion of Class I Odorant Receptors Impacts Perception of Carboxylic Acids.

The mammalian main olfactory pathway detects myriad volatile chemicals using >1,000 odorant receptor (OR) genes, which are organized into two phylogenetically distinct classes (class I and class II). An important question is how these evolutionarily conserved classes contribute to odor perception. Here, we report functional inactivation of a large number of class I ORs in mice via identification and deletion of a local cis-acting enhancer in the class I gene cluster. This manipulation reduced expression of half of the 131 intact class I genes. The resulting class I-depleted mice exhibited a significant reduction in the number of glomeruli responding to carboxylic acids-chemicals associated with microbial action and body odors. These mice also exhibit a change in odor perception marked by a selective loss of behavioral aversion to these compounds. Together, our data demonstrate that class I ORs play a critical role in representing a class of biologically relevant chemosignals.

Single olfactory receptors set odor detection thresholds.

In many species, survival depends on olfaction, yet the mechanisms that underlie olfactory sensitivity are not well understood. Here we examine how a conserved subset of olfactory receptors, the trace amine-associated receptors (TAARs), determine odor detection thresholds of mice to amines. We find that deleting all TAARs, or even single TAARs, results in significant odor detection deficits. This finding is not limited to TAARs, as the deletion of a canonical odorant receptor reduced behavioral sensitivity to its preferred ligand. Remarkably, behavioral threshold is set solely by the most sensitive receptor, with no contribution from other highly sensitive receptors. In addition, increasing the number of sensory neurons (and glomeruli) expressing a threshold-determining TAAR does not improve detection, indicating that sensitivity is not limited by the typical complement of sensory neurons. Our findings demonstrate that olfactory thresholds are set by the single highest affinity receptor and suggest that TAARs are evolutionarily conserved because they determine the sensitivity to a class of biologically relevant chemicals.

Cytoarchitecture of the telencephalon in the coral reef multiband Butterflyfish ( Chaetodon multicinctus : Perciformes).

Detailed neuroanatomical studies of model species are necessary to facilitate comparative experiments which test hypotheses relevant to brain evolution and function. Butterflyfishes (Chaetodontidae) boast numerous sympatric species that differ in social behavior, aggression and feeding ecology. However, the ability to test hypotheses relevant to brain function in this family is hindered by the lack of detailed neural descriptions. The cytoarchitecture of the telencephalon in the monogamous and territorial multiband butterflyfish, Chaetodon multicinctus, was determined with Nissl-stained serial sections and an immunohistochemical analysis of arginine vasotocin (AVT), serotonin, substance P and tyrosine hydroxylase. The ventral telencephalon was similar to that of other perciform fishes studied, with one major difference. A previously undescribed postcommissural region, the cuneate nucleus, was identified and putatively assigned to the ventral telencephalon. While the function of this nucleus is unknown, preliminary studies indicate that it may be part of a behaviorally relevant subpallial neural circuit that is modulated by AVT. The dorsal telencephalon consisted of 15 subdivisions among central, medial, lateral, dorsal and posterior zones. Several regions of the dorsal telencephalon of C. multicinctus differed from many other perciform fishes examined thus far. The nucleus taenia was in a more caudal position, and the central and lateral zones were enlarged. Within the lateral zone, an unusual third, ventral subdivision and a large-celled division were present. One hypothesis is that the enlarged ventral subdivision of the lateral zone (potential hippocampus homolog) relates to an enhancement of spatial learning or olfactory memory, which are important for this coral reef fish. This study provides the neuroanatomical basis for future comparative and evolutionary studies of brain organization and neuropeptide distributions, physiological studies of neural processing and insight into the complex social behavior of butterflyfishes.

Non-redundant coding of aversive odours in the main olfactory pathway.

Many species are critically dependent on olfaction for survival. In the main olfactory system of mammals, odours are detected by sensory neurons that express a large repertoire of canonical odorant receptors and a much smaller repertoire of trace amine-associated receptors (TAARs). Odours are encoded in a combinatorial fashion across glomeruli in the main olfactory bulb, with each glomerulus corresponding to a specific receptor. The degree to which individual receptor genes contribute to odour perception is unclear. Here we show that genetic deletion of the olfactory Taar gene family, or even a single Taar gene (Taar4), eliminates the aversion that mice display to low concentrations of volatile amines and to the odour of predator urine. Our findings identify a role for the TAARs in olfaction, namely, in the high-sensitivity detection of innately aversive odours. In addition, our data reveal that aversive amines are represented in a non-redundant fashion, and that individual main olfactory receptor genes can contribute substantially to odour perception.

Fast drum strokes: novel and convergent features of sonic muscle ultrastructure, innervation, and motor neuron organization in the Pyramid Butterflyfish (Hemitaurichthys polylepis).

Sound production that is mediated by intrinsic or extrinsic swim bladder musculature has evolved multiple times in teleost fishes. Sonic muscles must contract rapidly and synchronously to compress the gas-filled bladder with sufficient velocity to produce sound. Muscle modifications that may promote rapid contraction include small fiber diameter, elaborate sarcoplasmic reticulum (SR), triads at the A-I boundary, and cores of sarcoplasm. The diversity of innervation patterns indicate that sonic muscles have independently evolved from different trunk muscle precursors. The analysis of sonic motor pathways in distantly related fishes is required to determine the relationships between sonic muscle evolution and function in acoustic signaling. We examined the ultrastructure of sonic and adjacent hypaxial muscle fibers and the distribution of sonic motor neurons in the coral reef Pyramid Butterflyfish (Chaetodontidae: Hemitaurichthys polylepis) that produces sound by contraction of extrinsic sonic muscles near the anterior swim bladder. Relative to adjacent hypaxial fibers, sonic muscle fibers were sparsely arranged among the endomysium, smaller in cross-section, had longer sarcomeres, a more elaborate SR, wider t-tubules, and more radially arranged myofibrils. Both sonic and non-sonic muscle fibers possessed triads at the Z-line, lacked sarcoplasmic cores, and had mitochondria among the myofibrils and concentrated within the peripheral sarcoplasm. Sonic muscles of this derived eutelost possess features convergent with other distant vocal taxa (other euteleosts and non-euteleosts): small fiber diameter, a well-developed SR, and radial myofibrils. In contrast with some sonic fishes, however, Pyramid Butterflyfish sonic muscles lack sarcoplasmic cores and A-I triads. Retrograde nerve label experiments show that sonic muscle is innervated by central and ventrolateral motor neurons associated with spinal nerves 1-3. This restricted distribution of sonic motor neurons in the spinal cord differs from many euteleosts and likely reflects the embryological origin of sonic muscles from hypaxial trunk precursors rather than occipital somites.

Uncoupling stimulus specificity and glomerular position in the mouse olfactory system.

Sensory information is often mapped systematically in the brain with neighboring neurons responding to similar stimulus features. The olfactory system represents chemical information as spatial and temporal activity patterns across glomeruli in the olfactory bulb. However, the degree to which chemical features are mapped systematically in the glomerular array has remained controversial. Here, we test the hypothesis that the dual roles of odorant receptors, in axon guidance and odor detection, can serve as a mechanism to map olfactory inputs with respect to their function. We compared the relationship between response specificity and glomerular position in genetically-defined olfactory sensory neurons expressing variant odorant receptors. We find that sensory neurons with the same odor response profile can be mapped to different regions of the bulb, and that neurons with different response profiles can be mapped to the same glomeruli. Our data demonstrate that the two functions of odorant receptors can be uncoupled, indicating that the mechanisms that map olfactory sensory inputs to glomeruli do so without regard to stimulus specificity.

An olfactory subsystem that mediates high-sensitivity detection of volatile amines.

Olfactory stimuli are detected by over 1,000 odorant receptors in mice, with each receptor being mapped to specific glomeruli in the olfactory bulb. The trace amine-associated receptors (TAARs) are a small family of evolutionarily conserved olfactory receptors whose contribution to olfaction remains enigmatic. Here, we show that a majority of the TAARs are mapped to a discrete subset of glomeruli in the dorsal olfactory bulb of the mouse. This TAAR projection is distinct from the previously described class I and class II domains, and is formed by a sensory neuron population that is restricted to express TAAR genes prior to choice. We also show that the dorsal TAAR glomeruli are selectively activated by amines at low concentrations. Our data uncover a hard-wired, parallel input stream in the main olfactory pathway that is specialized for the detection of volatile amines.

Arginine vasotocin neuronal phenotypes and their relationship to aggressive behavior in the territorial monogamous multiband butterflyfish, Chaetodon multicinctus.

Intra and interspecific comparisons of arginine vasotocin (AVT) and its mammalian homolog arginine vasopressin (AVP) demonstrate several relationships between these neuropeptides and aggression/dominance behaviors. Prior studies in coral reef butterflyfishes and other fishes indicate that features of AVT neurons in the gigantocellular preoptic area (gPOA) and axon varicosities within the ventral nucleus of the ventral telencephalon should have a positive relationship with aggressive behavior, whereas AVT-ir neuronal features in the parvocellular preoptic area (pPOA) should have a negative relationship. We measured the offensive aggression of wild caught territorial monogamous multiband butterflyfish, Chaetodon multicinctus, in a simple lab paradigm that controlled for social context and variations in social stimuli. Offensive aggression did not follow a clear stereotyped pattern, but rather a complex sequence that includes five action patterns and two approach behaviors. We then used immunohistochemistry to test for associations between AVT immunoreactive features and projections with overall offensive aggression. Our results indicate that gPOA cell number was positively related to aggression while both the size and number of pPOA cells were negatively related to aggression. No association between aggression and the number of axon varicosities in the telencephalic region proposed to be associated with aggression was found. This study provides further support for the relationship between AVT neuronal features and aggression in fishes, and provides preliminary evidence that this relationship may relate to the motivation to produce aggressive behaviors in the immediate future.

Arginine vasotocin neuronal phenotypes, telencephalic fiber varicosities, and social behavior in butterflyfishes (Chaetodontidae): potential similarities to birds and mammals.

The neuropeptide arginine vasopressin (AVP) influences many social behaviors through its action in the forebrain of mammals. However, the function of the homologous arginine vasotocin (AVT) in the forebrain of fishes, specifically the telencephalon remains unresolved. We tested whether the density of AVT-immunoreactive (-ir) fiber varicosities, somata size or number of AVT-ir neuronal phenotypes within the forebrain were predictive of social behavior in reproductive males of seven species of butterflyfishes (family Chaetodontidae) in four phylogenetic clades. Similar to other fishes, the aggressive (often territorial) species in most cases had larger AVT-ir cells within the gigantocellular preoptic cell group. Linear discriminant function analyses demonstrated that the density of AVT-ir varicosities within homologous telencephalic nuclei to those important for social behavior in mammals and birds were predictive of aggressive behavior, social affiliations, and mating system. Of note, the density of AVT-ir varicosities within the ventral nucleus of the ventral telencephalon, thought to be homologous to the septum of other vertebrates, was the strongest predictor of aggressive behavior, social affiliation, and mating system. These results are consistent with the postulate that AVT within the telencephalon of fishes plays an important role in social behavior and may function in a similar manner to that of AVT/AVP in birds and mammals despite having cell populations solely within the preoptic area.