Persistent Neurological Deficits in Mouse PASC Reveal Antiviral Drug Limitations.

Post-Acute Sequelae of COVID-19 (PASC) encompasses persistent neurological symptoms, including olfactory and autonomic dysfunction. Here, we report chronic neurological dysfunction in mice infected with a virulent mouse-adapted SARS-CoV-2 that does not infect the brain. Long after recovery from nasal infection, we observed loss of tyrosine hydroxylase (TH) expression in olfactory bulb glomeruli and neurotransmitter levels in the substantia nigra (SN) persisted. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased levels of proinflammatory cytokines and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, as found in human neurodegenerative diseases. Early treatment with antivirals (nirmatrelvir and molnupiravir) reduced virus titers and lung inflammation but failed to prevent neurological abnormalities, as observed in patients. Together these results show that chronic deficiencies in neuronal function in SARS-CoV-2-infected mice are not directly linked to ongoing olfactory epithelium dysfunction. Rather, they bear similarity with neurodegenerative disease, the vulnerability of which is exacerbated by chronic inflammation.

Activity-dependent formation of the topographic map and the critical period in the development of mammalian olfactory system.

Neural activity influences every aspect of nervous system development. In olfactory systems, sensory neurons expressing the same odorant receptor project their axons to stereotypically positioned glomeruli, forming a spatial map of odorant receptors in the olfactory bulb. As individual odors activate unique combinations of glomeruli, this map forms the basis for encoding olfactory information. The establishment of this stereotypical olfactory map requires coordinated regulation of axon guidance molecules instructed by spontaneous activity. Recent studies show that sensory experiences also modify innervation patterns in the olfactory bulb, especially during a critical period of the olfactory system development. This review examines evidence in the field to suggest potential mechanisms by which various aspects of neural activity regulate axon targeting. We also discuss the precise functions served by neural plasticity during the critical period.

Spatiotemporal Mapping and Molecular Basis of Whole-brain Circuit Maturation.

Brain development is highly dynamic and asynchronous, marked by the sequential maturation of functional circuits across the brain. The timing and mechanisms driving circuit maturation remain elusive due to an inability to identify and map maturing neuronal populations. Here we create DevATLAS (Developmental Activation Timing-based Longitudinal Acquisition System) to overcome this obstacle. We develop whole-brain mapping methods to construct the first longitudinal, spatiotemporal map of circuit maturation in early postnatal mouse brains. Moreover, we uncover dramatic impairments within the deep cortical layers in a neurodevelopmental disorders (NDDs) model, demonstrating the utility of this resource to pinpoint when and where circuit maturation is disrupted. Using DevATLAS, we reveal that early experiences accelerate the development of hippocampus-dependent learning by increasing the synaptically mature granule cell population in the dentate gyrus. Finally, DevATLAS enables the discovery of molecular mechanisms driving activity-dependent circuit maturation.

Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2.

Spatial transcriptomics maps RNA molecules to the location in a tissue where they are expressed. Here we document the use of Slide-SeqV2 to visualize gene expression in the mouse olfactory bulb (OB). This approach relies on spatially identified beads to locate and quantify individual transcripts. The expression profiles associated with the beads are used to identify and localize individual cell types in an unbiased manner. We demonstrate the various cell types and subtypes with distinct spatial locations in the olfactory bulb that are identified using Slide-SeqV2.

Protocol to detect RNAs from tissue sections in mice using Y-branched probe in situ hybridization.

Here, we describe a fluorescent in situ hybridization protocol named Yn-situ, standing for Y-branched probe in situ hybridization, to detect RNAs from mice tissue sections. We provide steps for the synthesis and quantification of preamplifier probe using nickase. We also detail the preparation of tissue section, probe hybridization, signal development using hybridization chain reaction (HCR), and quantification of the signals. This approach avoids the use of proprietary in situ hybridization techniques, therefore reducing costs. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).

Robust and sensitive in situ RNA detection using Yn-situ.

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger a hybridization chain reaction (HCR) to detect polynucleotides. By characterizing the performance of this method, we show that the Yn-situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of RNA molecules using fewer probes targeting short nucleotide sequences than existing methods. A set of five probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNAscope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.

Maximal Dependence Capturing as a Principle of Sensory Processing.

Sensory inputs conveying information about the environment are often noisy and incomplete, yet the brain can achieve remarkable consistency in recognizing objects. Presumably, transforming the varying input patterns into invariant object representations is pivotal for this cognitive robustness. In the classic hierarchical representation framework, early stages of sensory processing utilize independent components of environmental stimuli to ensure efficient information transmission. Representations in subsequent stages are based on increasingly complex receptive fields along a hierarchical network. This framework accurately captures the input structures; however, it is challenging to achieve invariance in representing different appearances of objects. Here we assess theoretical and experimental inconsistencies of the current framework. In its place, we propose that individual neurons encode objects by following the principle of maximal dependence capturing (MDC), which compels each neuron to capture the structural components that contain maximal information about specific objects. We implement the proposition in a computational framework incorporating dimension expansion and sparse coding, which achieves consistent representations of object identities under occlusion, corruption, or high noise conditions. The framework neither requires learning the corrupted forms nor comprises deep network layers. Moreover, it explains various receptive field properties of neurons. Thus, MDC provides a unifying principle for sensory processing.

A physicochemical model of odor sampling.

We present a general physicochemical sampling model for olfaction, based on established pharmacological laws, in which arbitrary combinations of odorant ligands and receptors can be generated and their individual and collective effects on odor representations and olfactory performance measured. Individual odor ligands exhibit receptor-specific affinities and efficacies; that is, they may bind strongly or weakly to a given receptor, and can act as strong agonists, weak agonists, partial agonists, or antagonists. Ligands interacting with common receptors compete with one another for dwell time; these competitive interactions appropriately simulate the degeneracy that fundamentally defines the capacities and limitations of odorant sampling. The outcome of these competing ligand-receptor interactions yields a pattern of receptor activation levels, thereafter mapped to glomerular presynaptic activation levels based on the convergence of sensory neuron axons. The metric of greatest interest is the mean discrimination sensitivity, a measure of how effectively the olfactory system at this level is able to recognize a small change in the physicochemical quality of a stimulus. This model presents several significant outcomes, both expected and surprising. First, adding additional receptors reliably improves the system's discrimination sensitivity. Second, in contrast, adding additional ligands to an odorscene initially can improve discrimination sensitivity, but eventually will reduce it as the number of ligands increases. Third, the presence of antagonistic ligand-receptor interactions produced clear benefits for sensory system performance, generating higher absolute discrimination sensitivities and increasing the numbers of competing ligands that could be present before discrimination sensitivity began to be impaired. Finally, the model correctly reflects and explains the modest reduction in odor discrimination sensitivity exhibited by transgenic mice in which the specificity of glomerular targeting by primary olfactory neurons is partially disrupted.

Encoding innately recognized odors via a generalized population code.

Odors carrying intrinsic values often trigger instinctive aversive or attractive responses. It is not known how innate valence is encoded. An intuitive model suggests that the information is conveyed through specific channels in hardwired circuits along the olfactory pathway, insulated from influences of other odors, to trigger innate responses. Here, we show that in mice, mixing innately aversive or attractive odors with a neutral odor and, surprisingly, mixing two odors with the same valence, abolish the innate behavioral responses. Recordings from the olfactory bulb indicate that odors are not masked at the level of peripheral activation and glomeruli independently encode components in the mixture. In contrast, crosstalk among the mitral and tufted (M/T) cells changes their patterns of activity such that those elicited by the mixtures can no longer be linearly decoded as separate components. The changes in behavioral and M/T cell responses are associated with reduced activation of brain areas linked to odor preferences. Thus, crosstalk among odor channels at the earliest processing stage in the olfactory pathway leads to re-coding of odor identity to abolish valence associated with the odors. These results are inconsistent with insulated labeled lines and support a model of a common mechanism of odor recognition for both innate and learned valence associations.

Acquisition of innate odor preference depends on spontaneous and experiential activities during critical period.

Animals possess an inborn ability to recognize certain odors to avoid predators, seek food, and find mates. Innate odor preference is thought to be genetically hardwired. Here we report that acquisition of innate odor recognition requires spontaneous neural activity and is influenced by sensory experience during early postnatal development. Genetic silencing of mouse olfactory sensory neurons during the critical period has little impact on odor sensitivity, discrimination, and recognition later in life. However, it abolishes innate odor preference and alters the patterns of activation in brain centers. Exposure to innately recognized odors during the critical period abolishes the associated valence in adulthood in an odor-specific manner. The changes are associated with broadened projection of olfactory sensory neurons and expression of axon guidance molecules. Thus, a delicate balance of neural activity is needed during the critical period in establishing innate odor preference and convergent axon input is required to encode innate odor valence.

Matrix metalloprotease-mediated cleavage of neural glial-related cell adhesion molecules activates quiescent olfactory stem cells via EGFR.

Quiescent stem cells have been found in multiple adult organs, and activation of these stem cells is critical to the restoration of damaged tissues in response to injury or stress. Existing evidence suggests that extrinsic cues from the extracellular matrix or supporting cells of various stem cell niches may interact with intrinsic components to initiate stem cell differentiation, but the molecular and cellular mechanisms regulating their activation are not fully understood. In the present study, we find that olfactory horizontal basal cells (HBCs) are stimulated by neural glial-related cell adhesion molecules (NrCAMs). NrCAM activation requires matrix metalloproteases (MMPs) and epidermal growth factor receptors (EGFRs). Inhibiting MMP activity or EGFR activation not only blocks HBC proliferation in the cultured olfactory organoids, but also severely suppresses HBC proliferation in the olfactory epithelium following methimazole-induced injury, resulting in a delay of olfactory mucosa reconstitution and functional recovery of the injured mice. Both NrCAMs and EGFR are expressed by the HBCs and their expression increases upon injury. Our data indicate that MMP-mediated cleavage of NrCAMs serves as an autocrine or paracrine signal that activates EGFRs on HBCs to trigger HBC proliferation and differentiation to reconstruct the entire olfactory epithelium following injury.

Innate immune signaling in the olfactory epithelium reduces odorant receptor levels: modeling transient smell loss in COVID-19 patients.

Post-infectious anosmias typically follow death of olfactory sensory neurons (OSNs) with a months-long recovery phase associated with parosmias. While profound anosmia is the leading symptom associated with COVID-19 infection, many patients regain olfactory function within days to weeks without distortions. Here, we demonstrate that sterile induction of anti-viral type I interferon signaling in the mouse olfactory epithelium is associated with diminished odor discrimination and reduced odor-evoked local field potentials. RNA levels of all class I, class II, and TAAR odorant receptors are markedly reduced in OSNs in a non-cell autonomous manner. We find that people infected with COVID-19 rate odors with lower intensities and have odor discrimination deficits relative to people that tested negative for COVID-19. Taken together, we propose that inflammatory-mediated loss of odorant receptor expression with preserved circuit integrity accounts for the profound anosmia and rapid recovery of olfactory function without parosmias caused by COVID-19.

Alkaline phosphatase-based chromogenic and fluorescence detection method for BaseScope™ In Situ hybridization.

The BaseScope™ assay is a novel, highly sensitive RNA in situ hybridization (ISH) technique, allowing detection of short RNA sequences as well as discrimination between single-nucleotide alterations. Multiplexing BaseScope™ ISH with immunofluorescence assay has proven challenging because the diffusion of colorimetric dyes such as Fast Red in aqueous solutions degrades spatial resolution. In this study, we explore alkaline phosphatase-based fluorescent signal detection methods and integrate it with BaseScope™ RNA ISH. We found that Fast Blue BB/NAMP can be used in BaseScope™ ISH for signal detection. Additionally, we found that the calcium binding fluorochromes calcein and xylenol orange can be used to localize alkaline phosphatase activity in both immunohistochemistry (IHC) and BaseScope™ ISH assays. When applied to mouse brain and intestine tissue sections, we successfully detected circular RNA molecules and cell proliferation antigen Ki-67 proteins. This technological advance expanded the substrate selection of alkaline phosphatase-based BaseScope™ RNA ISH to allow researchers and clinical professionals accurately assess RNA targets with immunofluorescent multiplexing.

A Population of Navigator Neurons Is Essential for Olfactory Map Formation during the Critical Period.

In the developing brain, heightened plasticity during the critical period enables the proper formation of neural circuits. Here, we identify the "navigator" neurons, a group of perinatally born olfactory sensory neurons, as playing an essential role in establishing the olfactory map during the critical period. The navigator axons project circuitously in the olfactory bulb and traverse multiple glomeruli before terminating in perspective glomeruli. These neurons undergo a phase of exuberant axon growth and exhibit a shortened lifespan. Single-cell transcriptome analyses reveal distinct molecular signatures for the navigators. Extending their lifespan prolongs the period of exuberant growth and perturbs axon convergence. Conversely, a genetic ablation experiment indicates that, despite postnatal neurogenesis, only the navigators are endowed with the ability to establish a convergent map. The presence and the proper removal of the navigator neurons are both required to establish tight axon convergence into the glomeruli.

G protein γ subunit Gγ13 is essential for olfactory function and aggressive behavior in mice.

Most olfactory receptors in vertebrates are G protein-coupled receptors, whose activation by odorants initiates intracellular signaling cascades through heterotrimeric G proteins consisting of α, ß, and γ subunits. Abolishment of the α subunits such as Gαolf in the main olfactory epithelium and Gαi2 and Gαo in the vomeronasal organ resulted in anosmia and/or impaired behavioral responses. In this study, we report that a G protein γ subunit, Gγ13, is expressed in a spatiotemporal manner similar to those of Gαolf and Gαi2 in the olfactory system and vomeronasal organ, respectively. In addition, Gγ13 was found in the glomeruli of the main olfactory bulb but was largely absent in the glomeruli of the accessory olfactory bulb. Using the Cre-loxP system, the Gγ13's gene Gng13 was nullified in the mature olfactory sensory neurons and apical vomeronasal sensory neurons where the Cre recombinase was expressed under the promoter of the Omp gene for the olfactory marker protein. Immunohistochemistry indicated much reduced expression of Gγ13 in the apical vomeronasal epithelium of the mutant mice. Behavioral experiments showed that the frequency and duration of aggressive encounters in the male mutant mice were significantly lower than in WT male mice. Taken together, these data suggest that the Gγ13 subunit is a critical signaling component in both the main olfactory epithelium and apical vomeronasal epithelium, and it plays an essential role in odor-triggered social behaviors including male-male aggression.

Pronounced strain-specific chemosensory receptor gene expression in the mouse vomeronasal organ.

BACKGROUND: The chemosensory system plays an important role in orchestrating sexual behaviors in mammals. Pheromones trigger sexually dimorphic behaviors and different mouse strains exhibit differential responses to pheromone stimuli. It has been speculated that differential gene expression in the sensory organs that detect pheromones may underlie sexually-dimorphic and strain-specific responses to pheromone cues. RESULTS: We have performed transcriptome analyses of the mouse vomeronasal organ, a sensory organ recognizing pheromones and interspecies cues. We find little evidence of sexual dimorphism in gene expression except for Xist, an essential gene for X-linked gene inactivation. Variations in gene expression are found mainly among strains, with genes from immune response and chemosensory receptor classes dominating the list. Differentially expressed genes are concentrated in genomic hotspots enriched in these families of genes. Some chemosensory receptors show exclusive patterns of expression in different strains. We find high levels of single nucleotide polymorphism in chemosensory receptor pseudogenes, some of which lead to functionalized receptors. Moreover, we identify a number of differentially expressed long noncoding RNA species showing strong correlation or anti-correlation with chemoreceptor genes. CONCLUSIONS: Our analyses provide little evidence supporting sexually dimorphic gene expression in the vomeronasal organ that may underlie dimorphic pheromone responses. In contrast, we find pronounced variations in the expression of immune response related genes, vomeronasal and G-protein coupled receptor genes among different mouse strains. These findings raised the possibility that diverse strains of mouse perceive pheromone cues differently and behavioral difference among strains in response to pheromone may first arise from differential detection of pheromones. On the other hand, sexually dimorphic responses to pheromones more likely originate from dimorphic neural circuits in the brain than from differential detection. Moreover, noncoding RNA may offer a potential regulatory mechanism controlling the differential expression patterns.

Regeneration and rewiring of rodent olfactory sensory neurons.

The olfactory sensory neurons are the only neurons in the mammalian nervous system that not only regenerate naturally and in response to injury, but also project to specific targets in the brain. The stem cells in the olfactory epithelium commit to both neuronal and non-neuronal lineages depending on the environmental conditions. They provide a continuous supply of new neurons. A newly generated neuron must express a specific odorant receptor gene and project to a central target consist of axons expressing the same receptor type. Recent studies have provided insights into this highly regulated, complex process. However, the molecular mechanisms that determine the regenerative capacity of stem cells, and the ability of newly generated neurons in directing their axons toward specific targets, remain elusive. Here we review progresses and controversies in the field and offer testable models.

Intracellular chloride concentration of the mouse vomeronasal neuron.

BACKGROUND: The vomeronasal organ (VNO) is specialized in detecting pheromone and heterospecific cues in the environment. Recent studies demonstrate the involvement of multiple ion channels in VNO signal transduction, including the calcium-activated chloride channels (CACCs). Opening of CACCs appears to result in activation of VNO neuron through outflow of Cl(-) ions. However, the intracellular Cl(-) concentration remains undetermined. RESULTS: We used the chloride ion quenching dye, MQAE, to measure the intracellular Cl(-) concentration of VNO neuron in live VNO slices. The resting Cl(-) concentration in the VNO neurons is measured at 84.73 mM. Urine activation of the VNO neurons causes a drop in Cl(-) concentration, consistent with the notion of an efflux of Cl(-) to depolarize the cells. Similar observation is made for VNO neurons from mice with deletion of the transient receptor potential canonical channel 2 (TRPC2), which have a resting Cl(-) concentrations at 81 mM. CONCLUSIONS: The VNO neurons rest at high intracellular Cl(-) concentration, which can lead to depolarization of the cell when chloride channels open. These results also provide additional support of TRPC2-independent pathway of VNO activation.

An Olfactory Cilia Pattern in the Mammalian Nose Ensures High Sensitivity to Odors.

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those in weakly stimulated regions. Surprisingly, reduction in neuronal excitability or ablation of the olfactory G protein in OSNs does not alter the cilia length pattern, indicating that neither spontaneous nor odor-evoked activity is required for its establishment. Furthermore, the pattern is evident at birth, maintained into adulthood, and restored following pharmacologically induced degeneration of the olfactory epithelium, suggesting that it is intrinsically programmed. Intriguingly, type III adenylyl cyclase (ACIII), a key protein in olfactory signal transduction and ubiquitous marker for primary cilia, exhibits location-dependent gene expression levels, and genetic ablation of ACIII dramatically alters the cilia pattern. These findings reveal an intrinsically programmed configuration in the nose to ensure high sensitivity to odors.