A transcriptional rheostat couples past activity to future sensory responses.

Animals traversing different environments encounter both stable background stimuli and novel cues, which are thought to be detected by primary sensory neurons and then distinguished by downstream brain circuits. Here, we show that each of the ∼1,000 olfactory sensory neuron (OSN) subtypes in the mouse harbors a distinct transcriptome whose content is precisely determined by interactions between its odorant receptor and the environment. This transcriptional variation is systematically organized to support sensory adaptation: expression levels of more than 70 genes relevant to transforming odors into spikes continuously vary across OSN subtypes, dynamically adjust to new environments over hours, and accurately predict acute OSN-specific odor responses. The sensory periphery therefore separates salient signals from predictable background via a transcriptional rheostat whose moment-to-moment state reflects the past and constrains the future; these findings suggest a general model in which structured transcriptional variation within a cell type reflects individual experience.

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.

Perception of sniff phase in mouse olfaction.

Olfactory systems encode odours by which neurons respond and by when they respond. In mammals, every sniff evokes a precise, odour-specific sequence of activity across olfactory neurons. Likewise, in a variety of neural systems, ranging from sensory periphery to cognitive centres, neuronal activity is timed relative to sampling behaviour and/or internally generated oscillations. As in these neural systems, relative timing of activity may represent information in the olfactory system. However, there is no evidence that mammalian olfactory systems read such cues. To test whether mice perceive the timing of olfactory activation relative to the sniff cycle ('sniff phase'), we used optogenetics in gene-targeted mice to generate spatially constant, temporally controllable olfactory input. Here we show that mice can behaviourally report the sniff phase of optogenetically driven activation of olfactory sensory neurons. Furthermore, mice can discriminate between light-evoked inputs that are shifted in the sniff cycle by as little as 10 milliseconds, which is similar to the temporal precision of olfactory bulb odour responses. Electrophysiological recordings in the olfactory bulb of awake mice show that individual cells encode the timing of photoactivation in relation to the sniff in both the timing and the amplitude of their responses. Our work provides evidence that the mammalian olfactory system can read temporal patterns, and suggests that timing of activity relative to sampling behaviour is a potent cue that may enable accurate olfactory percepts to form quickly.

Precise detection of direct glomerular input duration by the olfactory bulb.

Sensory neuron input to the olfactory bulb (OB) was activated precisely for different durations with blue light in mice expressing channelrhodopsin-2 in olfactory sensory neurons. Behaviorally the mice discriminated differences of 10 ms in duration of direct glomerular activation. In addition, a subset of mitral/tufted cells in the OB of awake mice responded tonically therefore conveying information on stimulus duration. Our study provides evidence that duration of the input to glomeruli not synchronized to sniffing is detected. This potent cue may be used to obtain information on puffs in odor plumes.

Ultrasensitive detection of amines by a trace amine-associated receptor.

The mammalian main olfactory pathway detects volatile chemicals using two families of G-protein-coupled receptors: a large repertoire of canonical odorant receptors and a much smaller set of trace amine-associated receptors (TAARs). The TAARs are evolutionarily conserved in vertebrates, including humans, suggesting an indispensible role in olfaction. However, little is known about the functional properties of TAARs when expressed in native olfactory sensory neurons. Here we describe experiments using gene targeting, electrophysiology, and optical imaging to study the response properties of TAAR-expressing sensory neurons and their associated glomeruli in mice. We show that olfactory sensory neurons that express a subset of the TAAR repertoire are preferentially responsive to amines. In addition, neurons expressing specific TAARs, TAAR3 or TAAR4, are highly sensitive and are also broadly tuned-responding to structurally diverse amines. Surprisingly, we find that TAAR4 is exquisitely sensitive, with apparent affinities for a preferred ligand, phenylethylamine, rivaling those seen with mammalian pheromone receptors. We provide evidence that this unprecedented sensitivity is mediated via receptor coupling to the canonical odorant transduction cascade. The data suggest that the TAARs are evolutionarily retained in the olfactory receptor repertoire to mediate high-sensitivity detection of a biologically relevant class of odorous stimuli.

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.

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 expression of trace amine-associated receptors requires cooperative cis-acting enhancers.

Olfactory sensory neurons express a large family of odorant receptors (ORs) and a small family of trace amine-associated receptors (TAARs). While both families are subject to so-called singular expression (expression of one allele of one gene), the mechanisms underlying TAAR gene choice remain obscure. Here, we report the identification of two conserved sequence elements in the mouse TAAR cluster (T-elements) that are required for TAAR gene expression. We observed that cell-type-specific expression of a TAAR-derived transgene required either T-element. Moreover, deleting either element reduced or abolished expression of a subset of TAAR genes, while deleting both elements abolished olfactory expression of all TAARs in cis with the mutation. The T-elements exhibit several features of known OR enhancers but also contain highly conserved, unique sequence motifs. Our data demonstrate that TAAR gene expression requires two cooperative cis-acting enhancers and suggest that ORs and TAARs share similar mechanisms of singular expression.

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.

Effects of FGF14 and NaVß4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons.

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVß4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVß4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse.

Genetically encoded probes show great promise in permitting functional imaging of specified neuronal populations in the intact nervous system, yet their in vivo application has been limited. Here, we have targeted expression of synapto-pHluorin, a pH-sensitive protein that reports synaptic vesicle fusion, to olfactory sensory neurons in mouse. Synapto-pHluorin selectively labeled presynaptic terminals of sensory neurons in glomeruli of the olfactory bulb. Odorant stimulation evoked large-amplitude fluorescence increases that were localized to individual glomeruli in vivo, correlated with presynaptic calcium influx, graded with stimulus intensity, and stable over a period of days. Spatial patterns of odorant-activated glomeruli were distributed and did not change systematically with increasing carbon chain length, in contrast to the finely organized chemotopy that has been reported using other imaging methods. Targeted expression of synapto-pHluorin in mouse will permit the analysis of previously inaccessible neuronal populations and chronic imaging from genetically identified neurons in vivo.

Sparsened neuronal activity in an optogenetically activated olfactory glomerulus.

Glomeruli are the functional units of olfactory information processing but little remains known about their individual unit function. This is due to their widespread activation by odor stimuli. We expressed channelrhodopsin-2 in a single olfactory sensory neuron type, and used laser stimulation and simultaneous in vivo calcium imaging to study the responses of a single glomerulus to optogenetic stimulation. Calcium signals in the neuropil of this glomerulus were representative of the sensory input and nearly identical if evoked by intensity-matched odor and laser stimuli. However, significantly fewer glomerular layer interneurons and olfactory bulb output neurons (mitral cells) responded to optogenetic versus odor stimuli, resulting in a small and spatially compact optogenetic glomerular unit response. Temporal features of laser stimuli were represented with high fidelity in the neuropil of the glomerulus and the mitral cells, but not in interneurons. Increases in laser stimulus intensity were encoded by larger signal amplitudes in all compartments of the glomerulus, and by the recruitment of additional interneurons and mitral cells. No spatial expansion of the glomerular unit response was observed in response to stronger input stimuli. Our data are among the first descriptions of input-output transformations in a selectively activated olfactory glomerulus.

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.

Stimulus dependent diversity and stereotypy in the output of an olfactory functional unit.

Olfactory inputs are organized in an array of functional units (glomeruli), each relaying information from sensory neurons expressing a given odorant receptor to a small population of output neurons, mitral/tufted (MT) cells. MT cells respond heterogeneously to odorants, and how the responses encode stimulus features is unknown. We recorded in awake mice responses from "sister" MT cells that receive input from a functionally characterized, genetically identified glomerulus, corresponding to a specific receptor (M72). Despite receiving similar inputs, sister MT cells exhibit temporally diverse, concentration-dependent, excitatory and inhibitory responses to most M72 ligands. In contrast, the strongest known ligand for M72 elicits temporally stereotyped, early excitatory responses in sister MT cells, consistent across a range of concentrations. Our data suggest that information about ligand affinity is encoded in the collective stereotypy or diversity of activity among sister MT cells within a glomerular functional unit in a concentration-tolerant manner.

Odorant receptor expression defines functional units in the mouse olfactory system.

Odorant receptors (ORs) mediate the interaction of odorous compounds with olfactory sensory neurons (OSNs) and influence the guidance of OSN axons to synaptic targets in the olfactory bulb (OB). OSNs expressing the same OR send convergent axonal projections to defined glomeruli in the OB and are thought to share the same odorant response properties. This expectation of functional similarity has not been tested experimentally, because it has not been possible to determine reproducibly the response properties of OSNs that express defined ORs. Here, we applied calcium imaging to characterize the odorant response properties of single neurons from gene-targeted mice in which the green fluorescent protein is coexpressed with a particular OR. We show that the odorants acetophenone and benzaldehyde are agonists for the M71 OR and that M71-expressing neurons are functionally similar in their response properties across concentration. Replacing the M71 coding sequence with that of the rat I7 OR changes the stimulus response profiles of this genetically defined OSN population and concomitantly results in the formation of novel glomeruli in the OB. We further show that the mouse I7 OR imparts a particular response profile to OSNs regardless of the epithelial zone of expression. Our data provide evidence that ORs determine both odorant specificity and axonal convergence and thus direct functionally similar afferents to form particular glomeruli. They confirm and extend the notion that OR expression provides a molecular basis for the formation and arrangement of glomerular functional units.

Sensory neurobiology: demystifying the sick sense.

The vomeronasal organ, a sensory structure within the olfactory system, detects chemical signals that affect social and sexual behaviors and that elicit responses to predator odors. A recent study demonstrates that innate avoidance of sick conspecifics requires an intact vomeronasal organ, expanding the repertoire of biological functions known to be mediated by this olfactory subsystem.

Multiple perceptible signals from a single olfactory glomerulus.

Glomeruli are functional units in the olfactory system. The mouse olfactory bulb contains roughly 2,000 glomeruli, each receiving inputs from olfactory sensory neurons (OSNs) that express a specific odorant receptor gene. Odors typically activate many glomeruli in complex combinatorial patterns and it is unknown which features of neuronal activity in individual glomeruli contribute to odor perception. To address this, we used optogenetics to selectively activate single, genetically identified glomeruli in behaving mice. We found that mice could perceive the stimulation of a single glomerulus. Single-glomerulus stimulation was also detected on an intense odor background. In addition, different input intensities and the timing of input relative to sniffing were discriminated through one glomerulus. Our data suggest that each glomerulus can transmit odor information using identity, intensity and temporal coding cues. These multiple modes of information transmission may enable the olfactory system to efficiently identify and localize odor sources.

Distinct effects of leucine or a mixture of the branched-chain amino acids (leucine, isoleucine, and valine) supplementation on resistance to fatigue, and muscle and liver-glycogen degradation, in trained rats.

OBJECTIVE: The aim of this study was to evaluate the effects of the mixture of branched-chain amino acids (BCAAs) supplementation compared with leucine administered orally on muscle biochemical parameters of trained rats submitted to an exercise-induced protocol of glycogen depletion. METHODS: After 6 wk of swimming exercise, 8 wk-old (250 g, adult) male Wistar rats were randomly divided into three experimental groups (n = 8 per group): the mixture of BCAAs (BCAAs), leucine (LEU), and placebo (PLA). All groups were submitted to swimming exercise for 6 wk and supplemented with either the mixture of BCAAs, leucine, or placebo during the last week of training. At week 7 of the protocol, the rats were submitted to an intermittent, progressive swimming test until exhaustion and sacrificed. Muscle gastrocnemius and liver were depicted to determine total glycogen, tricarboxylic acid cycle (TCA) intermediates, and enzymatic activities. Statistical evaluation was performed by one-way analysis of variance with Tukey post hoc test. RESULTS: Both muscle and liver glycogen degradation ratio were significantly higher in the mixture of BCAAs group compared to the PLA group (P < 0.05) and the LEU group presented decreased liver glycogen degradation ratio compared with the mixture of BCAAs group (P < 0.05). Both muscle and liver glycogen content were significantly spared in the mixture of BCAAs and LEU groups compared to the PLA group (P < 0.01). A performance test demonstrated that LEU supplementation enhanced resistance to exhaustion compared to the mixture of BCAAs (P < 0.001), however, no difference was found when LEU supplementation was compared to PLA (P > 0.05) Muscle citrate content was significantly higher in the mixture of BCAAs group compared with the PLA group (P < 0.001). Muscle malate content was significantly elevated in the mixture of BCAAs group compared with both the PLA (P < 0.001) and LEU groups (P < 0.001). BCAT activity was significantly reduced in the mixture of BCAAs supplementation group compared with the LEU group (P < 0.001). CONCLUSION: Leucine supplementation improved performance compared with the mixture of BCAAs supplementation, sparing muscle glycogen stores despite the augmentation of some TCA intermediate concentrations on the left side of the TCA cycle.

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.

The Alzheimer's ß-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb.

BACKGROUND: The ß-secretase, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), is a prime therapeutic target for lowering cerebral ß-amyloid (Aß) levels in Alzheimer's disease (AD). Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1-/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs), a well-studied model of axon targeting in vivo. RESULTS: We bred BACE1-/- mice with gene-targeted mice in which GFP is expressed from the loci of two odorant-receptors (ORs), MOR23 and M72, and olfactory marker protein (OMP) to produce offspring that were heterozygous for MOR23-GFP, M72-GFP, or OMP-GFP and were either BACE1+/+ or BACE1-/-. BACE1-/- mice had olfactory bulbs (OBs) that were smaller and weighed less than OBs of BACE1+/+ mice. In wild-type mice, BACE1 was present in OSN axon terminals in OB glomeruli. In whole-mount preparations and tissue sections, many OB glomeruli from OMP-GFP; BACE1-/- mice were malformed compared to wild-type glomeruli. MOR23-GFP; BACE1-/- mice had an irregular MOR23 glomerulus that was innervated by randomly oriented, poorly fasciculated OSN axons compared to BACE1+/+ mice. Most importantly, M72-GFP; BACE1-/- mice exhibited M72 OSN axons that were mis-targeted to ectopic glomeruli, indicating impaired axon guidance in BACE1-/- mice. CONCLUSIONS: Our results demonstrate that BACE1 is required for the accurate targeting of OSN axons and the proper formation of glomeruli in the OB, suggesting a role for BACE1 in axon guidance. OSNs continually undergo regeneration and hence require ongoing axon guidance. Neurogenesis and the regeneration of neurons and axons occur in other adult populations of peripheral and central neurons that also require axon guidance throughout life. Therefore, BACE1 inhibitors under development for the treatment of AD may potentially cause axon targeting defects in these neuronal populations as well.