Adaptive olfactory circuitry restores function despite severe olfactory bulb degeneration.

The olfactory bulb (OB) is a critical component of mammalian olfactory neuroanatomy. Beyond being the first and sole relay station for olfactory information to the rest of the brain, it also contains elaborate stereotypical circuitry that is considered essential for olfaction. Indeed, substantial lesions of the OB in rodents lead to anosmia. Here, we examined the circuitry that underlies olfaction in a mouse model with severe developmental degeneration of the OB. These mice could perform odor-guided tasks and even responded normally to innate olfactory cues. Despite the near total loss of the OB, piriform cortices in these mice responded to odors, and its neural activity sufficed to decode odor identity. We found that sensory neurons express the full repertoire of olfactory receptors, and their axons project primarily to the rudiments of the OB but also, ectopically, to olfactory cortical regions. Within the OB, the number of principal neurons was greatly reduced, and the morphology of their dendrites was abnormal, extending over large regions within the OB. Glomerular organization was totally lost in the severe cases of OB degeneration and altered in the more conserved OBs. This study shows that olfactory functionality can be preserved despite reduced and aberrant circuitry that is missing many of the elements believed to be essential for olfaction, and it may explain reported retention of olfaction in humans with degenerated OBs.

Stability and flexibility of odor representations in the mouse olfactory bulb.

Dynamic changes in sensory representations have been basic tenants of studies in neural coding and plasticity. In olfaction, relatively little is known about the dynamic range of changes in odor representations under different brain states and over time. Here, we used time-lapse in vivo two-photon calcium imaging to describe changes in odor representation by mitral cells, the output neurons of the mouse olfactory bulb. Using anesthetics as a gross manipulation to switch between different brain states (wakefulness and under anesthesia), we found that odor representations by mitral cells undergo significant re-shaping across states but not over time within state. Odor representations were well balanced across the population in the awake state yet highly diverse under anesthesia. To evaluate differences in odor representation across states, we used linear classifiers to decode odor identity in one state based on training data from the other state. Decoding across states resulted in nearly chance-level accuracy. In contrast, repeating the same procedure for data recorded within the same state but in different time points, showed that time had a rather minor impact on odor representations. Relative to the differences across states, odor representations remained stable over months. Thus, single mitral cells can change dynamically across states but maintain robust representations across months. These findings have implications for sensory coding and plasticity in the mammalian brain.

Plasticity in auditory cortex during parenthood.

Most animals display robust parental behaviors that support the survival and well-being of their offspring. The manifestation of parental behaviors is accompanied by physiological and hormonal changes, which affect both the body and the brain for better care giving. Rodents exhibit a behavior called pup retrieval - a stereotyped sequence of perception and action - used to identify and retrieve their newborn pups back to the nest. Pup retrieval consists of a significant auditory component, which depends on plasticity in the auditory cortex (ACx). We review the evidence of neural changes taking place in the ACx of rodents during the transition to parenthood. We discuss how the plastic changes both in and out of the ACx support the encoding of pup vocalizations. Key players in the mechanism of this plasticity are hormones and experience, both of which have a clear dynamic signature during the transition to parenthood. Mothers, co caring females, and fathers have been used as models to understand parental plasticity at disparate levels of organization. Yet, common principles of cortical plasticity and the biological mechanisms underlying its involvement in parental behavior are just beginning to be unpacked.

Learning-Induced Odor Modulation of Neuronal Activity in Auditory Cortex.

Sensory cortices, even of primary regions, are not purely unisensory. Rather, cortical neurons in sensory cortex show various forms of multisensory interactions. While some multisensory interactions naturally co-occur, the combination of others will co-occur through experience. In real life, learning and experience will result in conjunction with seemingly disparate sensory information that ultimately becomes behaviorally relevant, impacting perception, cognition, and action. Here we describe a novel auditory discrimination task in mice, designed to manipulate the expectation of upcoming trials using olfactory cues. We show that, after learning, female mice display a transient period of several days during which they exploit odor-mediated expectations for making correct decisions. Using two-photon calcium imaging of single neurons in auditory cortex (ACx) during behavior, we found that the behavioral effects of odor-mediated expectations are accompanied by an odor-induced modulation of neuronal activity. Further, we find that these effects are manifested differentially, based on the response preference of individual cells. A significant portion of effects, but not all, are consistent with a predictive coding framework. Our data show that learning novel odor-sound associations evoke changes in ACx. We suggest that behaviorally relevant multisensory environments mediate contextual effects as early as ACx.SIGNIFICANCE STATEMENT Natural environments are composed of multisensory objects. It remains unclear whether and how animals learn the regularities of congruent multisensory associations and how these may impact behavior and neural activity. We tested how learned odor-sound associations affected single-neuron responses in auditory cortex. We introduce a novel auditory discrimination task for mice in which odors set different contexts of expectation to upcoming trials. We show that, although the task can be solved purely by sounds, odor-mediated expectation impacts performance. We further show that odors cause a modulation of neuronal activity in auditory cortex, which is correlated with behavior. These results suggest that learning prompts an interaction of odor and sound information as early as sensory cortex.

Surround suppression in mouse auditory cortex underlies auditory edge detection.

Surround suppression (SS) is a fundamental property of sensory processing throughout the brain. In the auditory system, the early processing stream encodes sounds using a one dimensional physical space-frequency. Previous studies in the auditory system have shown SS to manifest as bandwidth tuning around the preferred frequency. We asked whether bandwidth tuning can be found around frequencies away from the preferred frequency. We exploited the simplicity of spectral representation of sounds to study SS by manipulating both sound frequency and bandwidth. We recorded single unit spiking activity from the auditory cortex (ACx) of awake mice in response to an array of broadband stimuli with varying central frequencies and bandwidths. Our recordings revealed that a significant portion of neuronal response profiles had a preferred bandwidth that varied in a regular way with the sound's central frequency. To gain insight into the possible mechanism underlying these responses, we modelled neuronal activity using a variation of the "Mexican hat" function often used to model SS. The model accounted for response properties of single neurons with high accuracy. Our data and model show that these responses in ACx obey simple rules resulting from the presence of lateral inhibitory sidebands, mostly above the excitatory band of the neuron, that result in sensitivity to the location of top frequency edges, invariant to other spectral attributes. Our work offers a simple explanation for auditory edge detection and possibly other computations of spectral content in sounds.

The local and long-range input landscape of inhibitory neurons in mouse auditory cortex.

Roughly 20% of the neurons in the mouse cortex are inhibitory interneurons (INs). Of these, the three major subtypes are parvalbumin (PV), somatostatin (SST), and vasoactive intestinal polypeptide (VIP) expressing neurons. We used monosynaptic rabies tracing to compare the presynaptic input landscape onto these three IN subtypes in the mouse primary auditory cortex (A1). We compared both local patterns of monosynaptic inputs as well as long-range input patterns. The local monosynaptic input landscape to SST neurons was more widespread as compared to PV and VIP neurons. The brain-wide input landscape was rich and heterogeneous with >40 brain regions connecting to all the three INs subtypes from both hemispheres. The general pattern of the long-range input landscape was similar among the groups of INs. Nevertheless, a few differences could be identified. At low resolution, the proportion of local versus long-range inputs was smaller for PV neurons. At mesoscale resolution, we found fewer inputs from temporal association area to VIP INs, and more inputs to SST neurons from basal forebrain and lateral amygdala. Our work can be used as a resource for a quantitative comparison of the location and level of inputs impinging onto discrete populations of neurons in mouse A1.

MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B.

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.

Context-Dependent Inhibitory Control of Stimulus-Specific Adaptation.

Stimulus-specific adaptation (SSA) is the reduction in responses to frequent stimuli (standards) that does not generalize to rare stimuli (deviants). We investigated the contribution of inhibition in auditory cortex to SSA using two-photon targeted cell-attached recordings and optogenetic manipulations in male mice. We characterized the responses of parvalbumin (PV)-, somatostatin (SST)-, and vasoactive intestinal polypeptide (VIP)-expressing interneurons of layer 2/3, and of serotonin receptor 5HT3a-expressing interneurons of layer 1. All populations showed early-onset SSA. Unexpectedly, the PV, SST, and VIP populations exhibited a substantial late component of evoked activity, often stronger for standard than for deviant stimuli. Optogenetic suppression of PV neurons facilitated pyramidal neuron responses substantially more (approximately ×10) for deviants than for standards. VIP suppression decreased responses of putative PV neurons, specifically for standard but not for deviant stimuli. Thus, the inhibitory network does not generate cortical SSA, but powerfully controls its expression by differentially affecting the responses to deviants and to standards.SIGNIFICANCE STATEMENT Stimulus-specific adaptation (SSA) reflects the growing complexity of auditory processing along the ascending auditory system. In the presence of SSA, neuronal responses depend not only on the stimulus itself but also on the history of stimulation. Strong SSA in the fast, ascending auditory pathway first occurs in cortex. Here we studied the role of the cortical inhibitory network in shaping SSA, showing that while cortical inhibition does not generate SSA, it powerfully controls its expression. We deduce that the cortical network contributes in crucial ways to the properties of SSA.

Anesthesia in mice activates discrete populations of neurons throughout the brain.

The brain undergoes rapid, dramatic, and reversible transitioning between states of wakefulness and unconsciousness during natural sleep and in pathological conditions such as hypoxia, hypotension, and concussion. Transitioning can also be induced pharmacologically using general anesthetic agents. The effect is selective. Mobility, sensory perception, memory formation, and awareness are lost while numerous housekeeping functions persist. How is selective transitioning accomplished? Classically a handful of brainstem and diencephalic "arousal nuclei" have been implicated in driving brain-state transitions on the grounds that their net activity systematically varies with brain state. Here we used transgenic targeted recombination in active populations mice to label neurons active during wakefulness with one reporter and neurons active during pentobarbital-induced general anesthesia with a second, contrasting reporter. We found 'wake-on' and 'anesthesia-on' neurons in widely distributed regions-of-interest, but rarely encountered neurons labeled with both reporters. Nearly all labeled neurons were either wake-on or anesthesia-on. Thus, anesthesia-on neurons are not unique to the few nuclei discovered to date whose activity appears to increase during anesthesia. Rather neuronal populations selectively active during anesthesia are located throughout the brain where they likely play a causative role in transitioning between wakefulness and anesthesia. The widespread neuronal suppression reported in prior comparisons of the awake and anesthetized brain in animal models and noninvasive imaging in humans reflects only net differences. It misses the ubiquitous presence of neurons whose activity increases during anesthesia. The balance in recruitment of anesthesia-on versus wake-on neuronal populations throughout the brain may be a key driver of regional and global vigilance states. [Correction added on September 22, 2021, after first online publication: Due to a typesetting error, the abstract text was cut off. This has been corrected now.].

NDNF interneurons in layer 1 gain-modulate whole cortical columns according to an animal's behavioral state.

Processing of sensory information in neural circuits is modulated by an animal's behavioral state, but the underlying cellular mechanisms are not well understood. Focusing on the mouse visual cortex, here we analyze the role of GABAergic interneurons that are located in layer 1 and express Ndnf (L1 NDNF INs) in the state-dependent control over sensory processing. We find that the ongoing and sensory-evoked activity of L1 NDNF INs is strongly enhanced when an animal is aroused and that L1 NDNF INs gain-modulate local excitatory neurons selectively during high-arousal states by inhibiting their apical dendrites while disinhibiting their somata via Parvalbumin-expressing interneurons. Because active NDNF INs are evenly spread in L1 and can affect excitatory neurons across all cortical layers, this indicates that the state-dependent activation of L1 NDNF INs and the subsequent shift of inhibition in excitatory neurons toward their apical dendrites gain-modulate sensory processing in whole cortical columns.

Flexible categorization in the mouse olfactory bulb.

The ability to group sensory stimuli into categories is crucial for efficient interaction with a rich and ever-changing environment. In olfaction, basic features of categorical representation of odors were observed as early as in the olfactory bulb (OB). Categorical representation was described in mitral cells (MCs) as sudden transitions in responses to odors that were morphed along a continuum. However, it remains unclear to what extent such response dynamics actually reflect perceptual categories and decisions therein. Here, we tested the role of learning on category formation in the mouse OB, using in vivo two-photon calcium imaging and behavior. We imaged MC responses in naive mice and in awake behaving mice as they learned two tasks with different classification logic. In one task, a one-decision-boundary task, animals learned to classify odor mixtures based on the dominant compound in the mixtures. As expected, categorical representation of odors, which was evident already in naive animals, further increased following learning. In a second task, a multi-decision-boundary task, animals learned to classify odors independent of their chemical similarity. Here, odor discrimination was based on the meaning ascribed to them (either rewarding or not). Following the multi-decision-boundary task, odor representations by MCs reorganized according to the odor value in the new category. This functional reorganization was also reflected as a shift from predominantly excitatory odor responses to predominantly inhibitory odor responses. Our data show that odor representations by MCs are flexible, are shaped by task demands, and carry category-related information.

Auditory Corticothalamic Neurons Are Recruited by Motor Preparatory Inputs.

Corticothalamic (CT) neurons comprise the largest component of the descending sensory corticofugal pathway, but their contributions to brain function and behavior remain an unsolved mystery. To address the hypothesis that layer 6 (L6) CTs may be activated by extra-sensory inputs prior to anticipated sounds, we performed optogenetically targeted single-unit recordings and two-photon imaging of Ntsr1-Cre+ L6 CT neurons in the primary auditory cortex (A1) while mice were engaged in an active listening task. We found that L6 CTs and other L6 units began spiking hundreds of milliseconds prior to orofacial movements linked to sound presentation and reward, but not to other movements such as locomotion, which were not linked to an explicit behavioral task. Rabies tracing of monosynaptic inputs to A1 L6 CT neurons revealed a narrow strip of cholinergic and non-cholinergic projection neurons in the external globus pallidus, suggesting a potential source of motor-related input. These findings identify new pathways and local circuits for motor modulation of sound processing and suggest a new role for CT neurons in active sensing.

Author Correction: Functional organization and population dynamics in the mouse primary auditory cortex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Learning-related population dynamics in the auditory thalamus.

Learning to associate sensory stimuli with a chosen action involves a dynamic interplay between cortical and thalamic circuits. While the cortex has been widely studied in this respect, how the thalamus encodes learning-related information is still largely unknown. We studied learning-related activity in the medial geniculate body (MGB; Auditory thalamus), targeting mainly the dorsal and medial regions. Using fiber photometry, we continuously imaged population calcium dynamics as mice learned a go/no-go auditory discrimination task. The MGB was tuned to frequency and responded to cognitive features like the choice of the mouse within several hundred milliseconds. Encoding of choice in the MGB increased with learning, and was highly correlated with the learning curves of the mice. MGB also encoded motor parameters of the mouse during the task. These results provide evidence that the MGB encodes task- motor- and learning-related information.

The Temporal Association Cortex Plays a Key Role in Auditory-Driven Maternal Plasticity.

Mother-infant bonding develops rapidly following parturition and is accompanied by changes in sensory perception and behavior. Here, we study how ultrasonic vocalizations (USVs) are represented in the brain of mothers. Using a mouse line that allows temporally controlled genetic access to active neurons, we find that the temporal association cortex (TeA) in mothers exhibits robust USV responses. Rabies tracing from USV-responsive neurons reveals extensive subcortical and cortical inputs into TeA. A particularly dominant cortical source of inputs is the primary auditory cortex (A1), suggesting strong A1-to-TeA connectivity. Chemogenetic silencing of USV-responsive neurons in TeA impairs auditory-driven maternal preference in a pup-retrieval assay. Furthermore, dense extracellular recordings from awake mice reveal changes of both single-neuron and population responses to USVs in TeA, improving discriminability of pup calls in mothers compared with naive females. These data indicate that TeA plays a key role in encoding and perceiving pup cries during motherhood.

Barrel cortex VIP/ChAT interneurons suppress sensory responses in vivo.

Cortical interneurons expressing vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT) are sparsely distributed throughout the neocortex, constituting only 0.5% of its neuronal population. The co-expression of VIP and ChAT suggests that these VIP/ChAT interneurons (VChIs) can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). In vitro physiological studies quantified the response properties and local connectivity patterns of the VChIs; however, the function of VChIs has not been explored in vivo. To study the role of VChIs in cortical network dynamics and their long-range connectivity pattern, we used in vivo electrophysiology and rabies virus tracing in the barrel cortex of mice. We found that VChIs have a low spontaneous spiking rate (approximately 1 spike/s) in the barrel cortex of anesthetized mice; nevertheless, they responded with higher fidelity to whisker stimulation than the neighboring layer 2/3 pyramidal neurons (Pyrs). Analysis of long-range inputs to VChIs with monosynaptic rabies virus tracing revealed that direct thalamic projections are a significant input source to these cells. Optogenetic activation of VChIs in the barrel cortex of awake mice suppresses the sensory responses of excitatory neurons in intermediate amplitudes of whisker deflections while increasing the evoked spike latency. The effect of VChI activation on the response was similar for both high-whisking (HW) and low-whisking (LW) conditions. Our findings demonstrate that, despite their sparsity, VChIs can effectively modulate sensory processing in the cortical microcircuit.

The Pre-synaptic Landscape of Mitral/Tufted Cells of the Main Olfactory Bulb.

In olfaction, all volatile odor information is tunneled through the main olfactory bulb (OB). Odor information is then processed before it is transferred to higher brain centers. Odor processing in the OB is carried out by numerous local inhibitory circuits and modulated by top-down input. Top-down modulation of OB function has been shown to act via interneurons but evidence also exists for its direct impact onto the principle mitral and tufted cells (M/Ts). Here, we used monosynaptic rabies trans-synaptic tracing from the OB to map and quantify the local and top-down pre-synaptic landscape of M/Ts and local inhibitory interneurons. We found that M/Ts receive a significant amount of top-down inputs from various brain regions that match qualitatively but not quantitatively those that synapse onto local inhibitory inter-neurons. These results show that M/Ts are direct targets of top-down inputs.

Rapid Changes in Synaptic Strength After Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of Americans annually, but effective treatments remain inadequate due to our poor understanding of how injury impacts neural function. Data are particularly limited for mild, closed-skull TBI, which forms the majority of human cases, and for acute injury phases, when trauma effects and compensatory responses appear highly dynamic. Here we use a mouse model of mild TBI to characterize injury-induced synaptic dysfunction, and examine its progression over the hours to days after trauma. Mild injury consistently caused both locomotor deficits and localized neuroinflammation in piriform and entorhinal cortices, along with reduced olfactory discrimination ability. Using whole-cell recordings to characterize synaptic input onto piriform pyramidal neurons, we found moderate effects on excitatory or inhibitory synaptic function at 48 h after TBI and robust increase in excitatory inputs in slices prepared 1 h after injury. Excitatory increases predominated over inhibitory effects, suggesting that loss of excitatory-inhibitory balance is a common feature of both mild and severe TBI. Our data indicate that mild injury drives rapidly evolving alterations in neural function in the hours following injury, highlighting the need to better characterize the interplay between the primary trauma responses and compensatory effects during this early time period.

Neural Correlates of Learning Pure Tones or Natural Sounds in the Auditory Cortex.

Associative learning of pure tones is known to cause tonotopic map expansion in the auditory cortex (ACx), but the function this plasticity sub-serves is unclear. We developed an automated training platform called the "Educage," which was used to train mice on a go/no-go auditory discrimination task to their perceptual limits, for difficult discriminations among pure tones or natural sounds. Spiking responses of excitatory and inhibitory parvalbumin (PV+) L2/3 neurons in mouse ACx revealed learning-induced overrepresentation of the learned frequencies, as expected from previous literature. The coordinated plasticity of excitatory and inhibitory neurons supports a role for PV+ neurons in homeostatic maintenance of excitation-inhibition balance within the circuit. Using a novel computational model to study auditory tuning curves, we show that overrepresentation of the learned tones does not necessarily improve discrimination performance of the network to these tones. In a separate set of experiments, we trained mice to discriminate among natural sounds. Perceptual learning of natural sounds induced "sparsening" and decorrelation of the neural response, consequently improving discrimination of these complex sounds. This signature of plasticity in A1 highlights its role in coding natural sounds.

The Hard and Soft Wired Nature of the Olfactory Map.

Across the animal kingdom, odors are known as potent stimuli that directly steer behavior. In 2007, Hitoshi Sakano and colleagues used the power of mouse genetics to manipulate the odor map in the olfactory bulb. Elegant behavioral, anatomical, and physiological analyses revealed an apparent dichotomy in how the brain interprets the odor map. Their work paved a way to think of behavioral contingencies as part of early olfactory processing, highlighting innate and learned pathways.