Perturbations in neural circuits can provide mechanistic understanding of the neural correlates of behavior. In M71 transgenic mice with a "monoclonal nose", glomerular input patterns in the olfactory bulb are massively perturbed and olfactory behaviors are altered. To gain insights into how olfactory circuits can process such degraded inputs we characterized odor-evoked responses of olfactory bulb mitral cells and interneurons. Surprisingly, calcium imaging experiments reveal that mitral cell responses in M71 transgenic mice are largely normal, highlighting a remarkable capacity of olfactory circuits to normalize sensory input. In vivo whole cell recordings suggest that feedforward inhibition from olfactory bulb periglomerular cells can mediate this signal normalization. Together, our results identify inhibitory circuits in the olfactory bulb as a mechanistic basis for many of the behavioral phenotypes of mice with a "monoclonal nose" and highlight how substantially degraded odor input can be transformed to yield meaningful olfactory bulb output.

Odor memories are exceptionally robust and essential for animal survival. The olfactory (piriform) cortex has long been hypothesized to encode odor memories, yet the cellular substrates for olfactory learning and memory remain unknown. Here, using intersectional, cFos-based genetic manipulations ("Fos tagging"), we show that olfactory fear conditioning activates sparse and distributed ensembles of neurons in the mouse piriform cortex. We demonstrate that chemogenetic silencing of these Fos-tagged piriform ensembles selectively interferes with odor fear memory retrieval but does not compromise basic odor detection and discrimination. Furthermore, chemogenetic reactivation of piriform neurons that were Fos tagged during olfactory fear conditioning causes a decrease in exploratory behavior, mimicking odor-evoked fear memory recall. Together, our experiments identify specific ensembles of piriform neurons as critical components of an olfactory fear memory trace.

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion, and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

The odorant receptor (OR) genes constitute the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion, through an unknown mechanism that likely exploits positive and negative regulation. We devised a genetic strategy in mice to examine OR selection by determining the transcriptional activity of an exogenous promoter homologously integrated into an OR locus. Using the tetracycline-dependent transactivator responsive promoter (tet(o)), we observed that the OR locus imposes spatial and temporal constraints on tet(o)-driven transcription. Conditional expression experiments reveal a developmental change in the permissiveness of the locus. Further, expression of an OR transgene that suppresses endogenous ORs similarly represses the OR-integrated tet(o). Neurons homozygous for the tet(o)-modified allele demonstrate predominantly monoallelic expression, despite their potential to express both copies. These data reveal multiple axes of regulation, and support a model of initiation of OR choice limited by nonpermissive chromatin and maintained by repression of nonselected alleles.

Sparse coding can improve discrimination of sensory stimuli by reducing overlap between their representations. Two factors, however, can offset sparse coding's benefits: similar sensory stimuli have significant overlap and responses vary across trials. To elucidate the effects of these 2 factors, we analyzed odor responses in the fly and mouse olfactory regions implicated in learning and discrimination-the mushroom body (MB) and the piriform cortex (PCx). We found that neuronal responses fall along a continuum from extremely reliable across trials to extremely variable or stochastic. Computationally, we show that the observed variability arises from noise within central circuits rather than sensory noise. We propose this coding scheme to be advantageous for coarse- and fine-odor discrimination. More reliable cells enable quick discrimination between dissimilar odors. For similar odors, however, these cells overlap and do not provide distinguishing information. By contrast, more unreliable cells are decorrelated for similar odors, providing distinguishing information, though these benefits only accrue with extended training with more trials. Overall, we have uncovered a conserved, stochastic coding scheme in vertebrates and invertebrates, and we identify a candidate mechanism, based on variability in a winner-take-all (WTA) inhibitory circuit, that improves discrimination with training.

Borders are important as they demarcate developing tissue into distinct functional units. A key challenge is the discovery of mechanisms that can convert morphogen gradients into tissue borders. While mechanisms that produce ultrasensitive cellular responses provide a solution, how extracellular morphogens drive such mechanisms remains poorly understood. Here, we show how Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) pathways interact to generate ultrasensitivity and borders in the dorsal telencephalon. BMP and FGF signaling manipulations in explants produced border defects suggestive of cross inhibition within single cells, which was confirmed in dissociated cultures. Using mathematical modeling, we designed experiments that ruled out alternative cross inhibition mechanisms and identified a cross-inhibitory positive feedback (CIPF) mechanism, or "toggle switch", which acts upstream of transcriptional targets in dorsal telencephalic cells. CIPF explained several cellular phenomena important for border formation such as threshold tuning, ultrasensitivity, and hysteresis. CIPF explicitly links graded morphogen signaling in the telencephalon to switch-like cellular responses and has the ability to form multiple borders and scale pattern to size. These benefits may apply to other developmental systems.

Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell-type-specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of CaV2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.

Distributed circuits wherein connections between subcircuit components seem randomly distributed are common to the olfactory circuit, hippocampus, and cerebellum. In such circuits, activation patterns seem random too, showing no detectable spatial preference, and contrast with regions that have topographic connections between subcircuits and topographic activation patterns. Quantitative studies of topographic circuits in the neocortex have yielded common principles of organization. Whether distributed circuits share similar principles of organization is unknown because similar quantitative information is missing and understanding the way they encode information remains a challenge. We addressed these needs by providing a quantitative description of the mouse piriform cortex, a paleocortical distributed circuit that subserves olfaction. The quantitative information provided two insights. First, with a nearly parameter-free model of the olfactory circuit, we show that the piriform cortex robustly maintains odor information and discrimination ability present in the olfactory bulb. Second, the paleocortex is quantitatively different from the neocortex: it has a lower surface area density, which decreases from the anterior to posterior paleocortex contrasting with the uniform neuronal density of the neocortex. These insights might also apply to other distributed circuits.

The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we show that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription defines spatially constrained OR repertoires, among which one OR allele may be selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization preferentially eliminate from this "privileged" repertoire ORs with more dorsal expression destinations, which are ectopically transcribed in neuronal progenitors throughout the olfactory epithelium. Our experiments identify early transcription as an "epigenetic" contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

The ability to target subclasses of neurons with defined connectivity is crucial for uncovering neural circuit functions. The olfactory (piriform) cortex is thought to generate odour percepts and memories, and odour information encoded in piriform is routed to target brain areas involved in multimodal sensory integration, cognition and motor control. However, it remains unknown if piriform outputs are spatially organized, and if distinct output channels are delineated by different gene expression patterns. Here we identify genes selectively expressed in different layers of the piriform cortex. Neural tracing experiments reveal that these layer-specific piriform genes mark different subclasses of neurons, which project to distinct target areas. Interestingly, these molecular signatures of connectivity are maintained in reeler mutant mice, in which neural positioning is scrambled. These results reveal that a predictive link between a neuron's molecular identity and connectivity in this cortical circuit is determined independent of its spatial position.

Identifying shared quantitative features of a neural circuit across species is important for 3 reasons. Often expressed in the form of power laws and called scaling relationships [1, 2], they reveal organizational principles of circuits, make insights gleaned from model systems widely applicable, and explain circuit performance and function, e.g., visual circuits [3, 4]. The visual circuit is topographic [5, 6], wherein retinal neurons target and activate predictable spatial loci in primary visual cortex. The brain, however, contains many circuits, where neuronal targets and activity are unpredictable and distributed throughout the circuit, e.g., olfactory circuits, in which glomeruli (or mitral cells) in the olfactory bulb synapse with neurons distributed throughout the piriform cortex [7-10]. It is unknown whether such circuits, which we term distributed circuits, are scalable. To determine whether distributed circuits scale, we obtained quantitative descriptions of the olfactory bulb and piriform cortex in six mammals using stereology techniques and light microscopy. Two conserved features provide evidence of scalability. First, the number of piriform neurons n and bulb glomeruli g scale as n∼g3/2. Second, the average number of synapses between a bulb glomerulus and piriform neuron is invariant at one. Using theory and modeling, we show that these two features preserve the discriminatory ability and precision of odor information across the olfactory circuit. As both abilities depend on circuit size, manipulating size provides evolution with a way to adapt a species to its niche without designing developmental programs de novo. These principles might apply to other distributed circuits like the hippocampus.