The acquisition of olfactory information and its early processing in mammals are modulated by brain states through sniffing behavior and neural feedback. We imaged the spatiotemporal pattern of odor-evoked activity in a population of output neurons (mitral/tufted cells, MTCs) in the olfactory bulb (OB) of head-restrained mice expressing a genetically-encoded calcium indicator. The temporal dynamics of MTC population activity were relatively simple in anesthetized animals, but were highly variable in awake animals. However, the apparently irregular activity in awake animals could be predicted well using sniff timing measured externally, or inferred through fluctuations in the global responses of MTC population even without explicit knowledge of sniff times. The overall spatial pattern of activity was conserved across states, but odor responses had a diffuse spatial component in anesthetized mice that was less prominent during wakefulness. Multi-photon microscopy indicated that MTC lateral dendrites were the likely source of spatially disperse responses in the anesthetized animal. Our data demonstrate that the temporal and spatial dynamics of MTCs can be significantly modulated by behavioral state, and that the ensemble activity of MTCs can provide information about sniff timing to downstream circuits to help decode odor responses.

The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of social information in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

Rodents can learn from exposure to rewarding odors to make better and quicker decisions. The piriform cortex is thought to be important for learning complex odor associations; however, it is not understood exactly how it learns to remember discriminations between many, sometimes overlapping, odor mixtures. We investigated how odor mixtures are represented in the posterior piriform cortex (pPC) of mice while they learn to discriminate a unique target odor mixture against hundreds of nontarget mixtures. We find that a significant proportion of pPC neurons discriminate between the target and all other nontarget odor mixtures. Neurons that prefer the target odor mixture tend to respond with brief increases in firing rate at odor onset compared to other neurons, which exhibit sustained and/or decreased firing. We allowed mice to continue training after they had reached high levels of performance and find that pPC neurons become more selective for target odor mixtures as well as for randomly chosen repeated nontarget odor mixtures that mice did not have to discriminate from other nontargets. These single unit changes during overtraining are accompanied by better categorization decoding at the population level, even though behavioral metrics of mice such as reward rate and latency to respond do not change. However, when difficult ambiguous trial types are introduced, the robustness of the target selectivity is correlated with better performance on the difficult trials. Taken together, these data reveal pPC as a dynamic and robust system that can optimize for both current and possible future task demands at once.

Associative learning depends on contingency, the degree to which a stimulus predicts an outcome. Despite its importance, the neural mechanisms linking contingency to behavior remain elusive. Here we examined the dopamine activity in the ventral striatum - a signal implicated in associative learning - in a Pavlovian contingency degradation task in mice. We show that both anticipatory licking and dopamine responses to a conditioned stimulus decreased when additional rewards were delivered uncued, but remained unchanged if additional rewards were cued. These results conflict with contingency-based accounts using a traditional definition of contingency or a novel causal learning model (ANCCR), but can be explained by temporal difference (TD) learning models equipped with an appropriate inter-trial-interval (ITI) state representation. Recurrent neural networks trained within a TD framework develop state representations like our best 'handcrafted' model. Our findings suggest that the TD error can be a measure that describes both contingency and dopaminergic activity.

Odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that both the identity of activated neurons and the timing of their activity convey information about odors. However, whether and how downstream neurons decipher these temporal patterns remains unknown. We addressed this question by measuring the spiking activity of downstream neurons while optogenetically stimulating two foci in the olfactory bulb with varying relative timing in mice. We found that the overall spike rates of piriform cortex neurons (PCNs) were sensitive to the relative timing of activation. Posterior PCNs showed higher sensitivity to relative input times than neurons in the anterior piriform cortex. In contrast, olfactory bulb neurons rarely showed such sensitivity. Thus, the brain can transform a relative time code in the periphery into a firing rate-based representation in central brain areas, providing evidence for the relevance of a relative time-based code in the olfactory bulb.

Most neurogenesis in the mammalian brain is completed embryonically, but in certain areas the production of neurons continues throughout postnatal life. The functional properties of mature postnatally generated neurons often match those of their embryonically produced counterparts. However, we show here that in the olfactory bulb (OB), embryonic and postnatal neurogenesis produce functionally distinct subpopulations of dopaminergic (DA) neurons. We define two subclasses of OB DA neuron by the presence or absence of a key subcellular specialisation: the axon initial segment (AIS). Large AIS-positive axon-bearing DA neurons are exclusively produced during early embryonic stages, leaving small anaxonic AIS-negative cells as the only DA subtype generated via adult neurogenesis. These populations are functionally distinct: large DA cells are more excitable, yet display weaker and - for certain long-latency or inhibitory events - more broadly tuned responses to odorant stimuli. Embryonic and postnatal neurogenesis can therefore generate distinct neuronal subclasses, placing important constraints on the functional roles of adult-born neurons in sensory processing.

Within a single sniff, the mammalian olfactory system can decode the identity and concentration of odorants wafted on turbulent plumes of air. Yet, it must do so given access only to the noisy, dimensionally-reduced representation of the odor world provided by olfactory receptor neurons. As a result, the olfactory system must solve a compressed sensing problem, relying on the fact that only a handful of the millions of possible odorants are present in a given scene. Inspired by this principle, past works have proposed normative compressed sensing models for olfactory decoding. However, these models have not captured the unique anatomy and physiology of the olfactory bulb, nor have they shown that sensing can be achieved within the 100-millisecond timescale of a single sniff. Here, we propose a rate-based Poisson compressed sensing circuit model for the olfactory bulb. This model maps onto the neuron classes of the olfactory bulb, and recapitulates salient features of their connectivity and physiology. For circuit sizes comparable to the human olfactory bulb, we show that this model can accurately detect tens of odors within the timescale of a single sniff. We also show that this model can perform Bayesian posterior sampling for accurate uncertainty estimation. Fast inference is possible only if the geometry of the neural code is chosen to match receptor properties, yielding a distributed neural code that is not axis-aligned to individual odor identities. Our results illustrate how normative modeling can help us map function onto specific neural circuits to generate new hypotheses.

Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons.

Primary olfactory cortical areas receive direct input from the olfactory bulb, but also have extensive associational connections that have been mainly studied with classical anatomical methods. Here, we shed light on the functional properties of associational connections in the anterior and posterior piriform cortices (aPC and pPC) using optophysiological methods. We found that the aPC receives dense functional connections from the anterior olfactory nucleus (AON), a major hub in olfactory cortical circuits. The local recurrent connectivity within the aPC, long invoked in cortical autoassociative models, is sparse and weak. By contrast, the pPC receives negligible input from the AON, but has dense connections from the aPC as well as more local recurrent connections than the aPC. Finally, there are negligible functional connections from the pPC to aPC. Our study provides a circuit basis for a more sensory role for the aPC in odor processing and an associative role for the pPC.

The mitral-granule cell (MC-GC) reciprocal synapse is an important source of auto- and lateral-inhibition in the olfactory bulb (OB), and this local inhibition is critical for odor discrimination. We may gain insight into the role of MC autoinhibition in olfaction by correlating the functional development of the autoinhibition with the postnatal development of olfactory function. We have studied the functional development of the MC-GC reciprocal synapse using whole-cell patch-clamp recordings from MCs and GCs in acute OB slices from 3- to 30-day-old rats. The magnitude of dendrodendritic inhibition (DDI) measured by depolarizing a single MC and recording recurrent inhibition in the same cell increased up to the fifteenth day of life (P15), but dropped between P15 and P30. The initial increase and later decrease in DDI was echoed by a similar increase and decrease in the frequency of miniature inhibitory post-synaptic currents, suggesting an accompanying modulation in the number of synapses available to participate in DDI. The late decrease in DDI could also result, in part, from a decrease in GC excitability as well as an increase in relative contribution of N-methyl d-aspartate (NMDA) receptors to γ-amino butyric acid (GABA) release from GC synapses. Changes in release probability of GABAergic synapses are unlikely to account for the late reduction in DDI, although they might contribute to the early increase during development. Our results demonstrate that the functional MC-GC circuit evolves over development in a complex manner that may include both construction and elimination of synapses.

We investigated the roles of two Rab-family proteins, Rab3a and Rab5a, in hippocampal synaptic transmission using real-time fluorescence imaging. During synaptic activity, Rab3a dissociated from synaptic vesicles and dispersed into neighbouring axonal regions. Dispersion required calcium-dependent exocytosis and was complete before the entire vesicle pool turned over. In contrast, even prolonged synaptic activity produced limited dispersion of Rab5a. A GTPase-deficient mutant, Rab3a (Q81L), dispersed more slowly than wild-type Rab3a, and decreased the rate of exocytosis and the size of the recycling pool of vesicles. While overexpression of Rab3a did not affect vesicle recycling, overexpression of Rab5a reduced the recycling pool size by 50%. We propose that while Rab3a preferentially associates with recycling synaptic vesicles and modulates their trafficking, Rab5a is largely excluded from recycling vesicles.

Signaling by neurotrophins such as the brain-derived neurotrophic factor (BDNF) is known to modulate development of interneurons, but the circuit effects of this modulation remain unclear. Here, we examined the impact of deleting TrkB, a BDNF receptor, in parvalbumin-expressing (PV) interneurons on the balance of excitation and inhibition (E-I) in cortical circuits. In the mouse olfactory cortex, TrkB deletion impairs multiple aspects of PV neuronal function including synaptic excitation, intrinsic excitability, and the innervation pattern of principal neurons. Impaired PV cell function resulted in aberrant spiking patterns in principal neurons in response to stimulation of sensory inputs. Surprisingly, dampened PV neuronal function leads to a paradoxical decrease in overall excitability in cortical circuits. Our study demonstrates that, by modulating PV circuit plasticity and development, TrkB plays a critical role in shaping the evoked pattern of activity in a cortical network.

Estimating the pose of multiple animals is a challenging computer vision problem: frequent interactions cause occlusions and complicate the association of detected keypoints to the correct individuals, as well as having highly similar looking animals that interact more closely than in typical multi-human scenarios. To take up this challenge, we build on DeepLabCut, an open-source pose estimation toolbox, and provide high-performance animal assembly and tracking-features required for multi-animal scenarios. Furthermore, we integrate the ability to predict an animal's identity to assist tracking (in case of occlusions). We illustrate the power of this framework with four datasets varying in complexity, which we release to serve as a benchmark for future algorithm development.

The broadly-distributed, non-topographic projections to and from the olfactory cortex may suggest a flat, non-hierarchical organization in odor information processing. Layer 2 principal neurons in the anterior piriform cortex (APC) can be divided into 2 subtypes: semilunar (SL) and superficial pyramidal (SP) cells. Although it is known that SL and SP cells receive differential inputs from the olfactory bulb (OB), little is known about their projections to other olfactory regions. Here, we examined axonal projections of SL and SP cells using a combination of mouse genetics and retrograde labeling. Retrograde tracing from the OB or posterior piriform cortex (PPC) showed that the APC projects to these brain regions mainly through layer 2b cells, and dual-labeling revealed many cells extending collaterals to both target regions. Furthermore, a transgenic mouse line specifically labeling SL cells showed that they send profuse axonal projections to olfactory cortical areas, but not to the OB. These findings support a model in which information flow from SL to SP cells and back to the OB is mediated by a hierarchical feedback circuit, whereas both SL and SP cells broadcast information to higher olfactory areas in a parallel manner.

Microglia play key roles in regulating synapse development and refinement in the developing brain, but it is unknown whether they are similarly involved during adult neurogenesis. By transiently depleting microglia from the healthy adult mouse brain, we show that microglia are necessary for the normal functional development of adult-born granule cells (abGCs) in the olfactory bulb. Microglial depletion reduces the odor responses of developing, but not preexisting GCs in vivo in both awake and anesthetized mice. Microglia preferentially target their motile processes to interact with mushroom spines on abGCs, and when microglia are absent, abGCs develop smaller spines and receive weaker excitatory synaptic inputs. These results suggest that microglia promote the development of excitatory synapses onto developing abGCs, which may impact the function of these cells in the olfactory circuit.

In odorant-rich environments, animals must be able to detect specific odorants of interest against variable backgrounds. However, studies have found that both humans and rodents are poor at analyzing the components of odorant mixtures, suggesting that olfaction is a synthetic sense in which mixtures are perceived holistically. We found that mice could be easily trained to detect target odorants embedded in unpredictable and variable mixtures. To relate the behavioral performance to neural representation, we imaged the responses of olfactory bulb glomeruli to individual odors in mice expressing the Ca(2+) indicator GCaMP3 in olfactory receptor neurons. The difficulty of segregating the target from the background depended strongly on the extent of overlap between the glomerular responses to target and background odors. Our study indicates that the olfactory system has powerful analytic abilities that are constrained by the limits of combinatorial neural representation of odorants at the level of the olfactory receptors.

The vomeronasal organ (VNO) has a key role in mediating the social and defensive responses of many terrestrial vertebrates to species- and sex-specific chemosignals. More than 250 putative pheromone receptors have been identified in the mouse VNO, but the nature of the signals detected by individual VNO receptors has not yet been elucidated. To gain insight into the molecular logic of VNO detection leading to mating, aggression or defensive responses, we sought to uncover the response profiles of individual vomeronasal receptors to a wide range of animal cues. Here we describe the repertoire of behaviourally and physiologically relevant stimuli detected by a large number of individual vomeronasal receptors in mice, and define a global map of vomeronasal signal detection. We demonstrate that the two classes (V1R and V2R) of vomeronasal receptors use fundamentally different strategies to encode chemosensory information, and that distinct receptor subfamilies have evolved towards the specific recognition of certain animal groups or chemical structures. The association of large subsets of vomeronasal receptors with cognate, ethologically and physiologically relevant stimuli establishes the molecular foundation of vomeronasal information coding, and opens new avenues for further investigating the neural mechanisms underlying behaviour specificity.

The serotonergic raphe nuclei are involved in regulating brain states over timescales of minutes and hours. We examined more rapid effects of raphe activation on two classes of principal neurons in the mouse olfactory bulb, mitral and tufted cells, which send olfactory information to distinct targets. Brief stimulation of the raphe nuclei led to excitation of tufted cells at rest and potentiation of their odor responses. While mitral cells at rest were also excited by raphe activation, their odor responses were bidirectionally modulated, leading to improved pattern separation of odors. In vitro whole-cell recordings revealed that specific optogenetic activation of raphe axons affected bulbar neurons through dual release of serotonin and glutamate. Therefore, the raphe nuclei, in addition to their role in neuromodulation of brain states, are also involved in fast, sub-second top-down modulation similar to cortical feedback. This modulation can selectively and differentially sensitize or decorrelate distinct output channels.

Positive and negative associations acquired through olfactory experience are thought to be especially strong and long-lasting. The conserved direct olfactory sensory input to the ventral striatal olfactory tubercle (OT) and its convergence with dense dopaminergic input to the OT could underlie this privileged form of associative memory, but how this process occurs is not well understood. We imaged the activity of the two canonical types of striatal neurons, expressing D1- or D2-type dopamine receptors, in the OT at cellular resolution while mice learned odor-outcome associations ranging from aversive to rewarding. D1 and D2 neurons both responded to rewarding and aversive odors. D1 neurons in the OT robustly and bidirectionally represented odor valence, responding similarly to odors predicting similar outcomes regardless of odor identity. This valence representation persisted even in the absence of a licking response to the odors and in the absence of the outcomes, indicating a true transformation of odor sensory information by D1 OT neurons. In contrast, D2 neuronal representation of the odor-outcome associations was weaker, contingent on a licking response by the mouse, and D2 neurons were more selective for odor identity than valence. Stimulus valence coding in the OT was modality-sensitive, with separate sets of D1 neurons responding to odors and sounds predicting the same outcomes, suggesting that integration of multimodal valence information happens downstream of the OT. Our results point to distinct representation of identity and valence of odor stimuli by D1 and D2 neurons in the OT.

Inputs from olfactory sensory neuron (OSN) axons expressing the same type of odorant receptor (OR) converge in the glomerulus of the main olfactory bulb. A key marker of mature OSNs is olfactory marker protein (OMP), whose deletion has been associated with deficits in OSN signal transduction and odor discrimination. Here, we investigate glomerular odor responses and anatomical architecture in mice in which one or both alleles of OMP are replaced by the fluorescent synaptic activity reporter, synaptopHluorin. Functionally heterogeneous glomeruli, that is, ones with microdomains with distinct odor responses, are rare in OMP+/- mice, but occur frequently in OMP-/- mice. Genetic targeting of single ORs reveals that these microdomains arise from co-innervation of individual glomeruli by OSNs expressing different ORs. This glomerular mistargeting is locally restricted to a few glomerular diameters. Our studies document functional heterogeneity in sensory input within individual glomeruli and uncover its anatomical correlate, revealing an unexpected role for OMP in the formation and refinement of the glomerular map.