Combining a Breath-Synchronized Olfactometer with Brain Simulation to Study the Impact of Odors on Corticospinal Excitability and Effective Connectivity.

It is widely accepted that olfactory stimulation elicits motor behaviors, such as approaching pleasant odorants and avoiding unpleasant ones, in animals and humans. Recently, studies using electroencephalography and transcranial magnetic stimulation (TMS) have demonstrated a strong link between processing in the olfactory system and activity in the motor cortex in humans. To better understand the interactions between the olfactory and the motor systems and to overcome some of the previous methodological limitations, we developed a new method combining an olfactometer that synchronizes the random order presentation of odorants with different hedonic values and the TMS (single- and dual-coil) triggering with nasal breathing phases. This method allows probing the modulations of corticospinal excitability and effective ipsilateral connectivity between the dorsolateral prefrontal cortex and the primary motor cortex that could occur during pleasant and unpleasant odor perception. The application of this method will allow for objectively discriminating the pleasantness value of an odorant in a given participant, indicating the biological impact of the odorant on brain effective connectivity and excitability. In addition, this could pave the way for clinical investigations in patients with neurological or neuropsychiatric disorders who may exhibit odor hedonic alterations and maladaptive approach-avoidance behaviors.

Long-term olfactory enrichment promotes non-olfactory cognition, noradrenergic plasticity and remodeling of brain functional connectivity in older mice.

Brain functional and structural changes lead to cognitive decline during aging, but a high level of cognitive stimulation during life can improve cognitive performances in the older adults, forming the cognitive reserve. Noradrenaline has been proposed as a molecular link between environmental stimulation and constitution of the cognitive reserve. Taking advantage of the ability of olfactory stimulation to activate noradrenergic neurons of the locus coeruleus, we used repeated olfactory enrichment sessions over the mouse lifespan to enable the cognitive reserve buildup. Mice submitted to olfactory enrichment, whether started in early or late adulthood, displayed improved olfactory discrimination at late ages and interestingly, improved spatial memory and cognitive flexibility. Moreover, olfactory and non-olfactory cognitive performances correlated with increased noradrenergic innervation in the olfactory bulb and dorsal hippocampus. Finally, c-Fos mapping and connectivity analysis revealed task-specific remodeling of functional neural networks in enriched older mice. Long-term olfactory enrichment thus triggers structural noradrenergic plasticity and network remodeling associated with better cognitive aging and thereby forms a promising mouse model of the cognitive reserve buildup.

Age-related differences in perception and coding of attractive odorants in mice.

Hedonic perception deeply changes with aging, significantly impacting health and quality of life in elderly. In young adult mice, an odor hedonic signature is represented along the antero-posterior axis of olfactory bulb, and transferred to the olfactory tubercle and ventral tegmental area, promoting approach behavior. Here, we show that while the perception of unattractive odorants was unchanged in older mice (22 months), the appreciation of some but not all attractive odorants declined. Neural activity in the olfactory bulb and tubercle of older mice was consistently altered when attraction to pleasant odorants was impaired while maintained when the odorants kept their attractivity. Finally, in a self-stimulation paradigm, optogenetic stimulation of the olfactory bulb remained rewarding in older mice even without ventral tegmental area's response to the stimulation. Aging degrades behavioral and neural responses to some pleasant odorants but rewarding properties of olfactory bulb stimulation persisted, providing new insights into developing novel olfactory training strategies to elicit motivation even when the dopaminergic system is altered as observed in normal and/or neurodegenerative aging.

Influence of the human body odor compound HMHA on face perception.

Body odors convey information about the individuals, but the mechanisms are not fully understood yet. As far as human reproduction is concerned, molecules that are produced in sexually dimorphic amounts could be possible chemosignals. 3-hydroxy-3-methylhexanoic acid (HMHA) is one of them-more typical of men. Here, we investigated the possibility that the perception of gender and attractiveness in human faces could be implicitly influenced by this compound. Clearly feminine, ambiguous and clearly masculine faces were primed with an odor of HMHA, a control odor or air. Based on 100-ms face presentation, 40 raters had to identify the face's gender as quickly as possible and provide attractiveness evaluations. 3-hydroxy-3-methylhexanoic acid tended to be perceived as less pleasant and induced lower sniff duration in women compared with men. As to the effects of HMHA on face perception (vs. control conditions), we found that gender identification and the associated response time were unaffected by HMHA. Attractiveness of the faces, however, increased in presence of HMHA, but not in a sex-specific manner and only for unattractive faces with ambiguous gender. In sum, this study found no evidence in favor of a possible role of this sexually dimorphic compound in intrasexual competition nor in intersexual attraction.

Twenty-four-hour rhythmicities in disorders of consciousness are associated with a favourable outcome.

Fluctuations of consciousness and their rhythmicities have been rarely studied in patients with a disorder of consciousness after acute brain injuries. 24-h assessment of brain (EEG), behaviour (eye-opening), and circadian (clock-controlled hormones secretion from urine) functions was performed in acute brain-injured patients. The distribution, long-term predictability, and rhythmicity (circadian/ultradian) of various EEG features were compared with the initial clinical status, the functional outcome, and the circadian rhythmicities of behaviour and clock-controlled hormones. Here we show that more physiological and favourable patterns of fluctuations are associated with a higher 24 h predictability and sharp up-and-down shape of EEG switches, reminiscent of the Flip-Flop model of sleep. Multimodal rhythmic analysis shows that patients with simultaneous circadian rhythmicity for brain, behaviour, and hormones had a favourable outcome. Finally, both re-emerging EEG fluctuations and homogeneous 24-h cycles for EEG, eye-opening, and hormones appeared as surrogates for preserved functionality in brainstem and basal forebrain, which are key prognostic factors for later improvement. While the recovery of consciousness has previously been related to a high short-term complexity, we suggest in this exploratory study the importance of the high predictability of the 24 h long-term generation of brain rhythms and highlight the importance of circadian body-brain rhythms in awakening.

Episodic memory and recognition are influenced by cues' sensory modality: comparing odours, music and faces using virtual reality.

Most everyday experiences are multisensory, and all senses can trigger the conscious re-experience of unique personal events embedded in their specific spatio-temporal context. Yet, little is known about how a cue's sensory modality influences episodic memory, and which step of this process is impacted. This study investigated recognition and episodic memory across olfactory, auditory and visual sensory modalities in a laboratory-ecological task using a non-immersive virtual reality device. At encoding, participants freely and actively explored unique and rich episodes in a three-room house where boxes delivered odours, musical pieces and pictures of face. At retrieval, participants were presented with modality-specific memory cues and were told to 1) recognise encoded cues among distractors and, 2) go to the room and select the box in which they encountered them at encoding. Memory performance and response times revealed that music and faces outperformed odours in recognition memory, but that odours and faces outperformed music in evoking encoding context. Interestingly, correct recognition of music and faces was accompanied by more profound inspirations than correct rejection. By directly comparing memory performance across sensory modalities, our study demonstrated that despite limited recognition, odours are powerful cues to evoke specific episodic memory retrieval.

Distinct brain networks for remote episodic memory depending on content and emotional experience.

Memories of life episodes are the heart of individual stories. However, modelling episodic memory is a major challenge in both humans and animals when considering all its characteristics. As a consequence, the mechanisms that underlie the storage of old nontraumatic episodic memories remain enigmatic. Here, using a new task in rodents that models human episodic memory including odour/place/context components and applying advances behavioural and computational analyses, we show that rats form and recollect integrated remote episodic memories of two occasionally encountered complex episodes occurring in their daily life. Similar to humans, the information content and accuracy of memories vary across individuals and depend on the emotional relationship with odours experienced during the very first episode. We used cellular brain imaging and functional connectivity analyses, to find out the engrams of remote episodic memories for the first time. Activated brain networks completely reflect the nature and content of episodic memories, with a larger cortico-hippocampal network when the recollection is complete and with an emotional brain network related to odours that is critical in maintaining accurate and vivid memories. The engrams of remote episodic memories remain highly dynamic since synaptic plasticity processes occur during recall related to memory updates and reinforcement.

Identification of new behavioral parameters to assess odorant hedonic value in humans: A naturalistic approach.

BACKGROUND: When you smell an odorant, your first reaction will certainly be either I like it or I dislike it. This primary reaction is a reflection of what is called the "hedonic value" of the odor. Very often, this hedonic value dominates the olfactory percept, more than olfactory identification or intensity. This component of olfactory perception is of primary importance for guiding behavior: avoiding danger (the smell of smoke, gas, etc.), consuming food, or seduction. Olfactory hedonics can be assessed using a large number of methods in humans, including psychophysical measures, autonomic responses, measurement of facial expressions or peripheral nervous activity. All of these techniques have their limitations: subjectivity, invasiveness, need for expertise, etc. A NEW METHOD: The olfactory system is closely linked to the reward system, the role of which is to mediate motivated behavior. In this context, we propose that the capacity odorants have of recruiting the reward system and thus inducing motivated behavior can be used to identify new behavioral parameters to assess odor hedonic value in humans. RESULTS: We recorded freely moving human participants exploring odors emanating from flasks, and showed that five parameters linked to motivated behavior were closely linked to odor hedonics: speed of approach to the nose and withdrawal of the flask containing the odorant, distance between flask and nose, number of samplings, and withdrawal distance (maximal distance between nose and flask after odor sampling). CONCLUSIONS: We highlighted new non-verbal and non-invasive parameters to evaluate olfactory hedonics in humans based on the assessment of odor-motivated behavior.

Protocol of controlled odorant stimulation for reducing apnoeic episodes in premature newborns: a randomised open-label Latin-square study with independent evaluation of the main endpoint (PREMODEUR).

INTRODUCTION: Apnoea affects 85% of premature infants under 34 weeks of age and would be an important risk factor for subsequent neuropsychological disorders. Currently, premature children with life-threatening apnoeas receive stimulants such as methylxanthines (mainly, caffeine) or doxapram (an analeptic unlicensed in children under 15). However, these products have undesirable effects (hyperarousal, irritability, sleep disorders, tachycardia) and are not always effective because apnoea does persist in some premature newborns. Previous studies have indicated that odorant stimulation, a non-invasive intervention, may stimulate the respiratory rhythm. The objective of the present protocol is to reduce the occurrence of apnoeic episodes in premature newborns by controlled odorant stimulation added to current pharmacological treatments. METHODS AND ANALYSIS: The project is a randomised open-label Latin-square trial with independent evaluation of the main endpoint. It will include 60 preterm neonates from two university hospital neonatal intensive care units over 2 years (2021-2023). Each newborn will receive no (S0), sham (S1) or real olfactory stimulation (S2) in random order. During S2, three distinct odorants (mint, grapefruit and vanilla) will be delivered successively, in puffs, over 24 hours. Mint and grapefruit odours stimulate the main and the trigeminal olfactory pathways, whereas vanilla odour stimulates only the main olfactory pathway. A statistical analysis will compare the incidence of apnoeic episodes during S1 versus S2 using a mixed effects Poisson model. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Comité de Protection des Personnes Île-de-France XI (# 2017-AO13-50-53). The results will be disseminated through various scientific meetings, specialised peer-reviewed journals and, whenever possible, posted on appropriate public websites. TRIAL REGISTRATION NUMBER: NCT02851979; Pre-results.

Neural processing of the reward value of pleasant odorants.

Pleasant odorants are represented in the posterior olfactory bulb (pOB) in mice. How does this hedonic information generate odor-motivated behaviors? Using optogenetics, we report here that stimulating the representation of pleasant odorants in a sensory structure, the pOB, can be rewarding, self-motivating, and is accompanied by ventral tegmental area activation. To explore the underlying neural circuitry downstream of the olfactory bulb (OB), we use 3D high-resolution imaging and optogenetics and determine that the pOB preferentially projects to the olfactory tubercle, whose increased activity is related to odorant attraction. We further show that attractive odorants act as reinforcers in dopamine-dependent place preference learning. Finally, we extend those findings to humans, who exhibit place preference learning and an increase BOLD signal in the olfactory tubercle in response to attractive odorants. Thus, strong and persistent attraction induced by some odorants is due to a direct gateway from the pOB to the reward system.

Modulation of olfactory-driven behavior by metabolic signals: role of the piriform cortex.

Olfaction is one of the major sensory modalities that regulates food consumption and is in turn regulated by the feeding state. Given that the olfactory bulb has been shown to be a metabolic sensor, we explored whether the anterior piriform cortex (aPCtx)-a higher olfactory cortical processing area-had the same capacity. Using immunocytochemical approaches, we report the localization of Kv1.3 channel, glucose transporter type 4, and the insulin receptor in the lateral olfactory tract and Layers II and III of the aPCtx. In current-clamped superficial pyramidal (SP) cells, we report the presence of two populations of SP cells: glucose responsive and non-glucose responsive. Using varied glucose concentrations and a glycolysis inhibitor, we found that insulin modulation of the instantaneous and spike firing frequency are both glucose dependent and require glucose metabolism. Using a plethysmograph to record sniffing frequency, rats microinjected with insulin failed to discriminate ratiometric enantiomers; considered a difficult task. Microinjection of glucose prevented discrimination of odorants of different chain-lengths, whereas injection of margatoxin increased the rate of habituation to repeated odor stimulation and enhanced discrimination. These data suggest that metabolic signaling pathways that are present in the aPCtx are capable of neuronal modulation and changing complex olfactory behaviors in higher olfactory centers.

Non-imaged based method for matching brains in a common anatomical space for cellular imagery.

BACKGROUND: Cellular imagery using histology sections is one of the most common techniques used in Neuroscience. However, this inescapable technique has severe limitations due to the need to delineate regions of interest on each brain, which is time consuming and variable across experimenters. NEW METHOD: We developed algorithms based on a vectors field elastic registration allowing fast, automatic realignment of experimental brain sections and associated labeling in a brain atlas with high accuracy and in a streamlined way. Thereby, brain areas of interest can be finely identified without outlining them and different experimental groups can be easily analyzed using conventional tools. This method directly readjusts labeling in the brain atlas without any intermediate manipulation of images. RESULTS: We mapped the expression of cFos, in the mouse brain (C57Bl/6J) after olfactory stimulation or a non-stimulated control condition and found an increased density of cFos-positive cells in the primary olfactory cortex but not in non-olfactory areas of the odor-stimulated animals compared to the controls. COMPARISON WITH EXISTING METHOD(S): Existing methods of matching are based on image registration which often requires expensive material (two-photon tomography mapping or imaging with iDISCO) or are less accurate since they are based on mutual information contained in the images. Our new method is non-imaged based and relies only on the positions of detected labeling and the external contours of sections. CONCLUSIONS: We thus provide a new method that permits automated matching of histology sections of experimental brains with a brain reference atlas.

Opposite regulation of inhibition by adult-born granule cells during implicit versus explicit olfactory learning.

Both passive exposure and active learning through reinforcement enhance fine sensory discrimination abilities. In the olfactory system, this enhancement is thought to occur partially through the integration of adult-born inhibitory interneurons resulting in a refinement of the representation of overlapping odorants. Here, we identify in mice a novel and unexpected dissociation between passive and active learning at the level of adult-born granule cells. Specifically, while both passive and active learning processes augment neurogenesis, adult-born cells differ in their morphology, functional coupling and thus their impact on olfactory bulb output. Morphological analysis, optogenetic stimulation of adult-born neurons and mitral cell recordings revealed that passive learning induces increased inhibitory action by adult-born neurons, probably resulting in more sparse and thus less overlapping odor representations. Conversely, after active learning inhibitory action is found to be diminished due to reduced connectivity. In this case, strengthened odor response might underlie enhanced discriminability.

In vivo beta and gamma subthreshold oscillations in rat mitral cells: origin and gating by respiratory dynamics.

In mammals, olfactory bulb (OB) dynamics are paced by slow and fast oscillatory rhythms at multiple levels: local field potential, spike discharge, and/or membrane potential oscillations. Interactions between these levels have been well studied for the slow rhythm linked to animal respiration. However, less is known regarding rhythms in the fast beta (10-35 Hz) and gamma (35-100 Hz) frequency ranges, particularly at the membrane potential level. Using a combination of intracellular and extracellular recordings in the OB of freely breathing rats, we show that beta and gamma subthreshold oscillations (STOs) coexist intracellularly and are related to extracellular local field potential (LFP) oscillations in the same frequency range. However, they are differentially affected by changes in cell excitability and by odor stimulation. This leads us to suggest that beta and gamma STOs may rely on distinct mechanisms: gamma STOs would mainly depend on mitral cell intrinsic resonance, while beta STOs could be mainly driven by synaptic activity. In a second study, we find that STO occurrence and timing are constrained by the influence of the slow respiratory rhythm on mitral and tufted cells. First, respiratory-driven excitation seems to favor gamma STOs, while respiratory-driven inhibition favors beta STOs. Second, the respiratory rhythm is needed at the subthreshold level to lock gamma and beta STOs in similar phases as their LFP counterparts and to favor the correlation between STO frequency and spike discharge. Overall, this study helps us to understand how the interaction between slow and fast rhythms at all levels of OB dynamics shapes its functional output. NEW & NOTEWORTHY In the mammalian olfactory bulb of a freely breathing anesthetized rat, we show that both beta and gamma membrane potential fast oscillation ranges exist in the same mitral and tufted (M/T) cell. Importantly, our results suggest they have different origins and that their interaction with the slow subthreshold oscillation (respiratory rhythm) is a key mechanism to organize their dynamics, favoring their functional implication in olfactory bulb information processing.

Significance of sniffing pattern during the acquisition of an olfactory discrimination task.

Active sampling of olfactory environment consists of sniffing in rodents. The importance of sniffing dynamics is well established at the neuronal and behavioral levels. Patterns of sniffing have been shown to be modulated by the physicochemical properties of odorants, particularly concentration and sorption. Sniffing is also heavily impacted by higher processing related to the behavioral context, emotion and attentional demand. However, how the pattern of sniffing evolves over the course of learning of an experimental olfactory conditioning is still poorly understood. We tested this question by monitoring sniffing activity, using a whole-body plethysmograph, on rats performing a two-alternative choice odor discrimination task. We followed sniff variations at different learning stages (naïve, well-trained, expert). We found that during the acquisition of an odor discrimination task, rats acquired a global sniffing pattern, independent of the odor pair used. This pattern consists of a longer sampling duration, a higher sniffing frequency, and a larger amplitude. In parallel, subtle differences of sniffing between the two odors of a pair were also observed. This sniffing behavior was not only associated with a better and faster acquisition of the discrimination task but was also transferred to other odor sets and refined after a long-term pause so as to reduce the sampling duration and maintain a specific sniffing frequency. Our results provide additional arguments that sniffing is a complex sensorimotor act that is strongly affected by olfactory learning.

Topographical representation of odor hedonics in the olfactory bulb.

Hedonic value is a dominant aspect of olfactory perception. Using optogenetic manipulation in freely behaving mice paired with immediate early gene mapping, we demonstrate that hedonic information is represented along the antero-posterior axis of the ventral olfactory bulb. Using this representation, we show that the degree of attractiveness of odors can be bidirectionally modulated by local manipulation of the olfactory bulb's neural networks in freely behaving mice.

Viewing Olfactory Affective Responses Through the Sniff Prism: Effect of Perceptual Dimensions and Age on Olfactomotor Responses to Odors.

Sniffing, which is the active sampling of olfactory information through the nasal cavity, is part of the olfactory percept. It is influenced by stimulus properties, affects how an odor is perceived, and is sufficient (without an odor being present) to activate the olfactory cortex. However, many aspects of the affective correlates of sniffing behavior remain unclear, in particular the modulation of volume and duration as a function of odor hedonics. The present study used a wide range of odorants with contrasted hedonic valence to test: (1) which psychophysical function best describes the relationship between sniffing characteristics and odor hedonics (e.g., linear, or polynomial); (2) whether sniffing characteristics are sensitive to more subtle variations in pleasantness than simple pleasant-unpleasant contrast; (3) how sensitive sniffing is to other perceptual dimensions of odors such as odor familiarity or edibility; and (4) whether the sniffing/hedonic valence relationship is valid in other populations than young adults, such as the elderly. Four experiments were conducted, using 16-48 odorants each, and recruiting a total of 102 participants, including a group of elderly people. Results of the four experiments were very consistent in showing that sniffing was sensitive to subtle variations in unpleasantness but not to subtle variations in pleasantness, and that, the more unpleasant the odor, the more limited the spontaneous sampling of olfactory information through the nasal cavity (smaller volume, shorter duration). This also applied, although to a lesser extent, to elderly participants. Relationships between sniffing and other perceptual dimensions (familiarity, edibility) were less clear. It was concluded that sniffing behavior might be involved in adaptive responses protecting the subject from possibly harmful substances.

"What-Where-Which" Episodic Retrieval Requires Conscious Recollection and Is Promoted by Semantic Knowledge.

Episodic memory is defined as the conscious retrieval of specific past events. Whether accurate episodic retrieval requires a recollective experience or if a feeling of knowing is sufficient remains unresolved. We recently devised an ecological approach to investigate the controlled cued-retrieval of episodes composed of unnamable odors (What) located spatially (Where) within a visual context (Which context). By combining the Remember/Know procedure with our laboratory-ecological approach in an original way, the present study demonstrated that the accurate odor-evoked retrieval of complex and multimodal episodes overwhelmingly required conscious recollection. A feeling of knowing, even when associated with a high level of confidence, was not sufficient to generate accurate episodic retrieval. Interestingly, we demonstrated that the recollection of accurate episodic memories was promoted by odor retrieval-cue familiarity and describability. In conclusion, our study suggested that semantic knowledge about retrieval-cues increased the recollection which is the state of awareness required for the accurate retrieval of complex episodic memories.

Memory of occasional events in rats: individual episodic memory profiles, flexibility, and neural substrate.

In search for the mechanisms underlying complex forms of human memory, such as episodic recollection, a primary challenge is to develop adequate animal models amenable to neurobiological investigation. Here, we proposed a novel framework and paradigm that provides means to quantitatively evaluate the ability of rats to form and recollect a combined knowledge of what happened, where it happened, and when or in which context it happened (referred to as episodic-like memory) after a few specific episodes in situations as close as possible to a paradigm we recently developed to study episodic memory in humans. In this task, rats have to remember two odor-drink associations (what happened) encountered in distinct locations (where it happened) within two different multisensory enriched environments (in which context/occasion it happened), each characterized by a particular combination of odors and places. By analyzing licking behavior on each drinking port, we characterized quantitatively individual recollection profiles and showed that rats are able to incidentally form and recollect an accurate, long-term integrated episodic-like memory that can last ≥ 24 d after limited exposure to the episodes. Placing rats in a contextually challenging recollection situation at recall reveals the ability for flexible use of episodic memory as described in humans. We further report that reversible inactivation of the dorsal hippocampus during recall disrupts the animal's capacity to recollect the complete episodic memory. Cellular imaging of c-Fos and Zif268 brain activation reveals that episodic memory recollection recruits a specific, distributed network of hippocampal-prefrontal cortex structures that correlates with the accuracy of the integrated recollection performance.

Cellular and molecular cues of glucose sensing in the rat olfactory bulb.

In the brain, glucose homeostasis of extracellular fluid is crucial to the point that systems specifically dedicated to glucose sensing are found in areas involved in energy regulation and feeding behavior. Olfaction is a major sensory modality regulating food consumption. Nutritional status in turn modulates olfactory detection. Recently it has been proposed that some olfactory bulb (OB) neurons respond to glucose similarly to hypothalamic neurons. However, the precise molecular cues governing glucose sensing in the OB are largely unknown. To decrypt these molecular mechanisms, we first used immunostaining to demonstrate a strong expression of two neuronal markers of glucose-sensitivity, insulin-dependent glucose transporter type 4 (GLUT4), and sodium glucose co-transporter type 1 (SGLT1) in specific OB layers. We showed that expression and mapping of GLUT4 but not SGLT1 were feeding state-dependent. In order to investigate the impact of metabolic status on the delivery of blood-borne glucose to the OB, we measured extracellular fluid glucose concentration using glucose biosensors simultaneously in the OB and cortex of anesthetized rats. We showed that glucose concentration in the OB is higher than in the cortex, that metabolic steady-state glucose concentration is independent of feeding state in the two brain areas, and that acute changes in glycemic conditions affect bulbar glucose concentration alone. These data provide new evidence of a direct relationship between the OB and peripheral metabolism, and emphasize the importance of glucose for the OB network, providing strong arguments toward establishing the OB as a glucose-sensing organ.