Odor encoding by signals in the olfactory bulb.

To understand the operation of the olfactory system, it is essential to know how information is encoded in the olfactory bulb. We applied Shannon information theoretic methods to address this, with signals from up to 57 glomeruli simultaneously optically imaged from presynaptic inputs in glomeruli in the mouse dorsal (dOB) and lateral (lOB) olfactory bulb, in response to six exemplar pure chemical odors. We discovered that, first, the tuning of these signals from glomeruli to a set of odors is remarkably broad, with a mean sparseness of 0.83 and a mean signal correlation of 0.64. Second, both of these factors contribute to the low information that is available from the responses of even populations of many tens of glomeruli, which was only 1.35 bits across 33 glomeruli on average, compared with the 2.58 bits required to perfectly encode these six odors. Third, although there is considerable interest in the possibility of temporal encoding of stimulus including odor identity, the amount of information in the temporal aspects of the presynaptic glomerular responses was low (mean 0.11 bits) and, importantly, was redundant with respect to the information available from the rates. Fourth, the information from simultaneously recorded glomeruli asymptotes very gradually and nonlinearly, showing that glomeruli do not have independent responses. Fifth, the information from a population became available quite rapidly, within 100 ms of sniff onset, and the peak of the glomerular response was at 200 ms. Sixth, the information from the lOB was not additive with that of the dOB.NEW & NOTEWORTHY We report broad tuning and low odor information available across the lateral and dorsal bulb populations of glomeruli. Even though response latencies can be significantly predictive of stimulus identity, such contained very little information and none that was not redundant with information based on rate coding alone. Last, in line with the emerging notion of the important role of earliest stages of responses ("primacy"), we report a very rapid rise in information after each inhalation.

Spatiotemporal dynamics of odor responses in the lateral and dorsal olfactory bulb.

The mammalian olfactory bulb (OB) plays an essential role in odor processing during the perception of smell. Optical imaging of the OB has proven to be a key tool in elucidating the spatial odor mapping and temporal dynamics that underlie higher-order odor processing. Much is known about the activation of olfactory sensory neuron (OSN) glomerular responses in the dorsal olfactory bulb (dOB) during odor presentation. However, the dorsal bulb provides access to only approximately 25% of all glomeruli, and little is known about how the lateral bulb functions during this critical process. Here, we report, for the first time, simultaneous measurements of OSN glomerular activity from both the dOB and the lateral olfactory bulb (lOB), thus describing odor-specific spatial mapping and the temporal dynamics of olfactory input to both the dorsal and lateral bulb. Odor responses in the lateral bulb tended to be most prominent in the dorso-lateral (D-L) region. Lateral glomeruli became active in a dorso-ventral (D-V) sequence upon odor inhalation, unlike the anterio-posterior (A-P) activity wave typical of the dorsal glomeruli. Across the entire D-L bulb, the spatial organization of these dynamics can be explained neither by the purely mechanosensitive dynamics (to breathing clean air) nor by the response amplitudes across glomeruli. Instead, these dynamics can be explained by a combination of zonal receptor distributions, associated OB projections, and air flow paths across the epithelium upon inhalation. Remarkably, we also found that a subset of OSN glomeruli in the lOB was highly sensitive to extranasal air pressure changes, a response type that has not been reported in dorsal glomeruli.

Orthonasal versus retronasal glomerular activity in rat olfactory bulb by fMRI.

Odorants can reach olfactory receptor neurons (ORNs) by two routes: orthonasally, when volatiles enter the nasal cavity during inhalation/sniffing, and retronasally, when food volatiles released in the mouth pass into the nasal cavity during exhalation/eating. Previous work in humans has shown that both delivery routes of the same odorant can evoke distinct perceptions and patterns of neural responses in the brain. Each delivery route is known to influence specific responses across the dorsal region of the glomerular sheet in the olfactory bulb (OB), but spatial distributions across the entire glomerular sheet throughout the whole OB remain largely unexplored. We used functional MRI (fMRI) to measure and compare activations across the entire glomerular sheet in rat OB resulting from both orthonasal and retronasal stimulations of the same odors. We observed reproducible fMRI activation maps of the whole OB during both orthonasal and retronasal stimuli. However, retronasal stimuli required double the orthonasal odor concentration for similar response amplitudes. Regardless, both the magnitude and spatial extent of activity were larger during orthonasal versus retronasal stimuli for the same odor. Orthonasal and retronasal response patterns show overlap as well as some route-specific dominance. Orthonasal maps were dominant in dorsal-medial regions, whereas retronasal maps were dominant in caudal and lateral regions. These different whole OB encodings likely underlie differences in odor perception between these biologically important routes for odorants among mammals. These results establish the relationships between orthonasal and retronasal odor representations in the rat OB.

Algorithms for Olfactory Search across Species.

Localizing the sources of stimuli is essential. Most organisms cannot eat, mate, or escape without knowing where the relevant stimuli originate. For many, if not most, animals, olfaction plays an essential role in search. While microorganismal chemotaxis is relatively well understood, in larger animals the algorithms and mechanisms of olfactory search remain mysterious. In this symposium, we will present recent advances in our understanding of olfactory search in flies and rodents. Despite their different sizes and behaviors, both species must solve similar problems, including meeting the challenges of turbulent airflow, sampling the environment to optimize olfactory information, and incorporating odor information into broader navigational systems.

Spontaneous activity forms a foundation for odor-evoked activation maps in the rat olfactory bulb.

Fluctuations in spontaneous activity have been observed by many neuroimaging techniques, but because these resting-state changes are not evoked by stimuli, it is difficult to determine how they relate to task-evoked activations. We conducted multi-modal neuroimaging scans of the rat olfactory bulb, both with and without odor, to examine interaction between spontaneous and evoked activities. Independent component analysis of spontaneous fluctuations revealed resting-state networks, and odor-evoked changes revealed activation maps. We constructed simulated activation maps using resting-state networks that were highly correlated to evoked activation maps. Simulated activation maps derived by intrinsic optical signal (IOS), which covers the dorsal portion of the glomerular sheet, significantly differentiated one odor's evoked activation map from the other two. To test the hypothesis that spontaneous activity of the entire glomerular sheet is relevant for representing odor-evoked activations, we used functional magnetic resonance imaging (fMRI) to map the entire glomerular sheet. In contrast to the IOS results, the fMRI-derived simulated activation maps significantly differentiated all three odors' evoked activation maps. Importantly, no evoked activation maps could be significantly differentiated using simulated activation maps produced using phase-randomized resting-state networks. Given that some highly organized resting-state networks did not correlate with any odors' evoked activation maps, we posit that these resting-state networks may characterize evoked activation maps associated with odors not studied. These results emphasize that fluctuations in spontaneous activity form a foundation for active processing, signifying the relevance of resting-state mapping to functional neuroimaging.

Perception of odors linked to precise timing in the olfactory system.

While the timing of neuronal activity in the olfactory bulb (OB) relative to sniffing has been the object of many studies, the behavioral relevance of timing information generated by patterned activation within the bulbar response has not been explored. Here we show, using sniff-triggered, dynamic, 2-D, optogenetic stimulation of mitral/tufted cells, that virtual odors that differ by as little as 13 ms are distinguishable by mice. Further, mice are capable of discriminating a virtual odor movie based on an optically imaged OB odor response versus the same virtual odor devoid of temporal dynamics-independently of the sniff-phase. Together with studies showing the behavioral relevance of graded glomerular responses and the response timing relative to odor sampling, these results imply that the mammalian olfactory system is capable of very high transient information transmission rates.

Comparison of glomerular activity patterns by fMRI and wide-field calcium imaging: Implications for principles underlying odor mapping.

Functional imaging signals arise from distinct metabolic and hemodynamic events at the neuropil, but how these processes are influenced by pre- and post-synaptic activities need to be understood for quantitative interpretation of stimulus-evoked mapping data. The olfactory bulb (OB) glomeruli, spherical neuropil regions with well-defined neuronal circuitry, can provide insights into this issue. Optical calcium-sensitive fluorescent dye imaging (OICa(2+)) reflects dynamics of pre-synaptic input to glomeruli, whereas high-resolution functional magnetic resonance imaging (fMRI) using deoxyhemoglobin contrast reveals neuropil function within the glomerular layer where both pre- and post-synaptic activities contribute. We imaged odor-specific activity patterns of the dorsal OB in the same anesthetized rats with fMRI and OICa(2+) and then co-registered the respective maps to compare patterns in the same space. Maps by each modality were very reproducible as trial-to-trial patterns for a given odor, overlapping by ~80%. Maps evoked by ethyl butyrate and methyl valerate for a given modality overlapped by ~80%, suggesting activation of similar dorsal glomerular networks by these odors. Comparison of maps generated by both methods for a given odor showed ~70% overlap, indicating similar odor-specific maps by each method. These results suggest that odor-specific glomerular patterns by high-resolution fMRI primarily tracks pre-synaptic input to the OB. Thus combining OICa(2+) and fMRI lays the framework for studies of OB processing over a range of spatiotemporal scales, where OICa(2+) can feature the fast dynamics of dorsal glomerular clusters and fMRI can map the entire glomerular sheet in the OB.

Testing the sorption hypothesis in olfaction: a limited role for sniff strength in shaping primary odor representations during behavior.

The acquisition of sensory information during behavior shapes the neural representation, central processing, and perception of external stimuli. In mammals, a sniff represents the basic unit of odor sampling, yet how sniffing shapes odor representations remains poorly understood. Perhaps the earliest hypothesis of the role of sniffing in olfaction arises from the fact that odorants with different physicochemical properties exhibit different patterns of deposition across the olfactory epithelium, and that these patterns are differentially affected by flow rate. However, whether sniff flow rates shape odor representations during natural sniffing remains untested, and whether animals make use of odorant sorption-airflow relationships as part of an active odor-sampling strategy remains unclear. We tested these ideas in the intact rat using a threefold approach. First, we asked whether sniff strength shapes odor representations in vivo by imaging from olfactory receptor neuron (ORN) terminals during controlled changes in inhalation flow in the anesthetized rat. Second, we asked whether sniff strength shapes odor representations by imaging from ORNs during natural sniffing in the awake rat. Third, we asked whether rats actively modulate sniff strength during an odor discrimination task. We found that, while artificial changes in flow rate can alter ORN responses, sniff strength has negligible effect on odor representations during natural sniffing, and behaving rats do not modulate flow rate to improve odor discrimination. These data suggest that modulating sniff strength does not shape odor representations sufficiently to be part of a strategy for active odor sensing in the behaving animal.

Retronasal odor representations in the dorsal olfactory bulb of rats.

Animals perceive their olfactory environment not only from odors originating in the external world (orthonasal route) but also from odors released in the oral cavity while eating food (retronasal route). Retronasal olfaction is crucial for the perception of food flavor in humans. However, little is known about the retronasal stimulus coding in the brain. The most basic questions are if and how route affects the odor representations at the level of the olfactory bulb (OB), where odor quality codes originate. We used optical calcium imaging of presynaptic dorsal OB responses to odorants in anesthetized rats to ask whether the rat OB could be activated retronasally, and how these responses compare to orthonasal responses under similar conditions. We further investigated the effects of specific odorant properties on orthonasal versus retronasal response patterns. We found that at a physiologically relevant flow rate, retronasal odorants can effectively reach the olfactory receptor neurons, eliciting glomerular response patterns that grossly overlap with those of orthonasal responses, but differ from the orthonasal patterns in the response amplitude and temporal dynamics. Interestingly, such differences correlated well with specific odorant properties. Less volatile odorants yielded relatively smaller responses retronasally, but volatility did not affect relative temporal profiles. More polar odorants responded with relatively longer onset latency and time to peak retronasally, but polarity did not affect relative response magnitudes. These data provide insight into the early stages of retronasal stimulus coding and establish relationships between orthonasal and retronasal odor representations in the rat OB.

Spatiotemporal features of neurovascular (un)coupling with stimulus-induced activity and hypercapnia challenge in cerebral cortex and olfactory bulb.

Carbon dioxide (CO2) is traditionally considered as metabolic waste, yet its regulation is critical for brain function. It is well accepted that hypercapnia initiates vasodilation, but its effect on neuronal activity is less clear. Distinguishing how stimulus- and CO2-induced vasodilatory responses are (dis)associated with neuronal activity has profound clinical and experimental relevance. We used an optical method in mice to simultaneously image fluorescent calcium (Ca2+) transients from neurons and reflectometric hemodynamic signals during brief sensory stimuli (i.e., hindpaw, odor) and CO2 exposure (i.e., 5%). Stimuli-induced neuronal and hemodynamic responses swiftly increased within locally activated regions exhibiting robust neurovascular coupling. However, hypercapnia produced slower global vasodilation which was temporally uncoupled to neuronal deactivation. With trends consistent across cerebral cortex and olfactory bulb as well as data from GCaMP6f/jRGECO1a mice (i.e., green/red Ca2+ fluorescence), these results unequivocally reveal that stimuli and CO2 generate comparable vasodilatory responses but contrasting neuronal responses. In summary, observations of stimuli-induced regional neurovascular coupling and CO2-induced global neurovascular uncoupling call for careful appraisal when using CO2 in gas mixtures to affect vascular tone and/or neuronal excitability, because CO2 is both a potent vasomodulator and a neuromodulator.

Thalamic activations in rat brain by fMRI during tactile (forepaw, whisker) and non-tactile (visual, olfactory) sensory stimulations.

The thalamus is a crucial subcortical hub that impacts cortical activity. Tracing experiments in animals and post-mortem humans suggest rich morphological specificity of the thalamus. Very few studies reported rodent thalamic activations by functional MRI (fMRI) as compared to cortical activations for different sensory stimuli. Here, we show different portions of the rat thalamus in response to tactile (forepaw, whisker) and non-tactile (visual, olfactory) sensory stimuli with high field fMRI (11.7T) using a custom-build quadrature surface coil to capture high sensitivity signals from superficial and deep brain regions simultaneously. Results demonstrate reproducible thalamic activations during both tactile and non-tactile stimuli. Forepaw and whisker stimuli activated broader regions within the thalamus: ventral posterior lateral (VPL), ventral posterior medial (VPM), lateral posterior mediorostral (LPMR) and posterior medial (POm) thalamic nuclei. Visual stimuli activated dorsal lateral geniculate nucleus (DLG) of the thalamus but also parts of the superior/inferior colliculus, whereas olfactory stimuli activated specifically the mediodorsal nucleus of the thalamus (MDT). BOLD activations in LGN and MDT were much stronger than in VPL, VPM, LPMR and POm. These fMRI-based thalamic activations suggest that forepaw and whisker (i.e., tactile) stimuli engage VPL, VPM, LPMR and POm whereas visual and olfactory (i.e., non-tactile) stimuli, respectively, recruit DLG and MDT exclusively.

Fluorescently-tagged magnetic protein nanoparticles for high-resolution optical and ultra-high field magnetic resonance dual-modal cerebral angiography.

Extremely small paramagnetic iron oxide nanoparticles (FeMNPs) (<5 nm) can enhance positive magnetic resonance imaging (MRI) contrast by shortening the longitudinal relaxation time of water (T1), but these nanoparticles experience rapid renal clearance. Here, magnetic protein nanoparticles (MPNPs) are synthesized from protein-conjugated citric acid coated FeMNPs (c-FeMNPs) without loss of the T1 MRI properties and tagged with fluorescent dye (f-MPNPs) for optical cerebrovascular imaging. The c-FeMNPs shows average size 3.8 ± 0.7 nm with T1 relaxivity (r1) of 1.86 mM-1 s-1 and transverse/longitudinal relaxivity ratio (r2/r1) of 2.53 at 11.7 T. The f-MPNPs show a higher r1 value of 2.18 mM-1 s-1 and r2/r1 ratio of 2.88 at 11.7 T, which generates excellent positive MRI contrast. In vivo cerebral angiography with f-MPNPs enables detailed microvascular contrast enhancement for differentiation of major blood vessels of murine brain, which corresponds well with whole brain three-dimensional time-of-flight MRI angiograms (17 min imaging time with 60 ms repetition time and 40 µm isotropic voxels). The real-time fluorescence angiography enables unambiguous detection of brain capillaries with diameter < 40 µm. Biodistribution examination revealed that f-MPNPs were safely cleared by the organs like the liver, spleen, and kidneys within a day after injection. Blood biochemical assays demonstrated no risk of iron overload in both rats and mice. With hybrid neuroimaging technologies (e.g., MRI-optical) on the rise, f-MPNPs built on this platform can generate exciting neuroscience applications.

Rapid encoding and perception of novel odors in the rat.

To gain insight into which parameters of neural activity are important in shaping the perception of odors, we combined a behavioral measure of odor perception with optical imaging of odor representations at the level of receptor neuron input to the rat olfactory bulb. Instead of the typical test of an animal's ability to discriminate two familiar odorants by exhibiting an operant response, we used a spontaneously expressed response to a novel odorant-exploratory sniffing-as a measure of odor perception. This assay allowed us to measure the speed with which rats perform spontaneous odor discriminations. With this paradigm, rats discriminated and began responding to a novel odorant in as little as 140 ms. This time is comparable to that measured in earlier studies using operant behavioral readouts after extensive training. In a subset of these trials, we simultaneously imaged receptor neuron input to the dorsal olfactory bulb with near-millisecond temporal resolution as the animal sampled and then responded to the novel odorant. The imaging data revealed that the bulk of the discrimination time can be attributed to the peripheral events underlying odorant detection: receptor input arrives at the olfactory bulb 100-150 ms after inhalation begins, leaving only 50-100 ms for central processing and response initiation. In most trials, odor discrimination had occurred even before the initial barrage of receptor neuron firing had ceased and before spatial maps of activity across glomeruli had fully developed. These results suggest a coding strategy in which the earliest-activated glomeruli play a major role in the initial perception of odor quality, and place constraints on coding and processing schemes based on simple changes in spike rate.

Sniffing controls an adaptive filter of sensory input to the olfactory bulb.

Most sensory stimuli are actively sampled, yet the role of sampling behavior in shaping sensory codes is poorly understood. Mammals sample odors by sniffing, a complex behavior that controls odorant access to receptor neurons. Whether sniffing shapes the neural code for odors remains unclear. We addressed this question by imaging receptor input to the olfactory bulb of awake rats performing odor discriminations that elicited different sniffing behaviors. High-frequency sniffing of an odorant attenuated inputs encoding that odorant, whereas lower sniff frequencies caused little attenuation. Odorants encountered later in a sniff bout were encoded as the combination of that odorant and the background odorant during low-frequency sniffing, but were encoded as the difference between the two odorants during high-frequency sniffing. Thus, sniffing controls an adaptive filter for detecting changes in the odor landscape. These data suggest an unexpected functional role for sniffing and show that sensory codes can be transformed by sampling behavior alone.

Evidence that the sweetness of odors depends on experience in rats.

Humans describe their perception of certain odorants in terms of taste qualities (e.g., sweet). It has also been found that in humans, novel odorants can rapidly and irreversibly acquire a taste, even after just a single pairing with a taste. It remains unclear whether flavor objects in general, and odor-taste generalizations in particular, are experience-dependent. Interactions might result from a failure by humans to sufficiently analyze the olfactory and gustatory components of compound flavorants. Here, we tested odor-taste generalizations in rats with or without paired exposure to an odorant and a tastant. We evaluated the generalization of conditioned odor aversion to tastants by rats. Our findings suggest that rats behave toward putatively tasteless retronasal odorants as if they were sweet only after prior paired experience of the odorant with a sweet tastant. These data support the hypothesis that taste-like qualities of odors are learned and are not innate. Furthermore, the present results suggest that acquisition of a taste quality by an odor need not depend on higher cognitive abilities. This study helps to establish the rat as a model for the study of behavioral neuroscience of flavor.

An automated sensitive approach for measuring whole gut transit time.

BACKGROUND: Commonly used methods to measure whole gut transit time in rodents have yet to combine high sensitivity, objectivity, and automation. We have developed a novel method using oral gavage of non-toxic fluorescent dye particles and their detection by fluorescence imaging to enable unbiased automated detection of gut transit time simultaneously in 8 cages. METHODS: Naïve mice (n = 20) were gavaged with a non-caloric viscous suspension of 4.4% fluorescent dye in 3 groups on 2 occasions. Each group was imaged in 8 cages at 5-minute intervals using blue LEDs for illumination and a Sony full-frame mirrorless camera with a green band-pass emission filter. Custom MATLAB code counted the number of fluorescent boli per cage post hoc and provided graphical and spreadsheet output. Boli counts across a wide range of parameters were compared to blind assessments by an experimenter. RESULTS: Fluorescent boli were detected with high sensitivity, while unstained boli were readily rejected. All cages showed no fluorescent boli for the first ~20 frames (100 minutes), after which many cages gradually show a rise to 1-6 fluorescent boli. The mean time to first fluorescent bolus in each session was 264 ± 141 and 223 ± 81 minutes post-gavage, with no within subject consistency. There was high correlation between automated scores and that of experimenter (r = .95 ± .02), being robust to parameter changes. CONCLUSIONS AND INFERENCES: This novel approach provides a reliable, automatic, and low-cost method of measuring gastrointestinal transit time in mice.

The neurocognitive bases of human multimodal food perception: consciousness.

This review explores how we become aware of the (integrated) flavor of food. In recent years, progress has been made understanding the neural correlates of consciousness. Experimental and computational data have been largely based on the visual system. Contemporary neurobiological frameworks of consciousness are reviewed, concluding that neural reverberation among forward- and back-projecting neural ensembles across brain areas is a common theme. In an attempt to extrapolate these concepts to the oral-sensory and olfactory systems involved with multimodal flavor perception, the integration of the sensory information of which into a flavor gestalt has been reviewed elsewhere (Verhagen, J.V., Engelen, L., 2006. The neurocognitive bases of human multimodal food perception: Sensory integration. Neurosci. Biobehav. Rev. 30(5): 613_650), I reconceptualize the flavor-sensory system by integrating it into a larger neural system termed the Homeostatic Interoceptive System (HIS). This system consists of an oral (taste, oral touch, etc.) and non-oral part (non oral-thermosensation, pain, etc.) which are anatomically and functionally highly similar. Consistent with this new concept and with a large volume of experimental data, I propose that awareness of intraoral food is related to the concomitant reverberant self-sustained activation of a coalition of neuronal subsets in agranular insula and orbitofrontal cortex (affect, hedonics) and agranular insula and perirhinal cortex (food identity), as well as the amygdala (affect and identity) in humans. I further discuss the functional anatomy in relation essential nodes. These formulations are by necessity to some extent speculative.

The neurocognitive bases of human multimodal food perception: sensory integration.

This review addresses a fundamental neuroscientific question in food perception: how multimodal features of food are integrated. Much research and conceptualization has emerged related to multisensory integration in vision, audition and somatosensation, while it remains poorly understood and researched within the chemical and mouth feel senses. This review aims to bridge this gap. We discuss the main concepts in the fields of auditory, visual and somatosensory multisensory integration and relate them to oral-sensory (gustatory and somatosensory) and olfactory (orolfactory) interactions. We systematically review the psychophysical literature pertaining to intra- and intermodal interactions related to food perception, while making explicit distinctions between peripheral and central interactions. As the neural bases of crossmodal orolfaction currently are poorly understood, we introduce several plausible neuroscientific models, which provide a framework for further neuroscientific exploration in this area. We are guided by a new meta-analysis of the odor-taste neuroimaging literature, as well as by single-unit, anatomical and psychophysical studies. Finally, we propose strong involvement of recurrent neural networks in multisensory integration and make suggestions for future research.