Murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors.

During social interactions, an individual's behavior is largely governed by the subset of signals emitted by others. Discrimination of "self" from "other" regulates the territorial urine countermarking behavior of mice. To identify the cues for this social discrimination and understand how they are interpreted, we designed an olfactory-dependent countermarking assay. We find major urinary proteins (MUPs) sufficient to elicit countermarking, and unlike other vomeronasal ligands that are detected by specifically tuned sensory neurons, MUPs are detected by a combinatorial strategy. A chemosensory signature of "self" that modulates behavior is developed via experience through exposure to a repertoire of MUPs. In contrast, aggression can be elicited by MUPs in an experience-independent but context-dependent manner. These findings reveal that individually emitted chemical cues can be interpreted based on their combinatorial permutation and relative ratios, and they can transmit both fixed and learned information to promote multiple behaviors.

Fast odour dynamics are encoded in the olfactory system and guide behaviour.

Odours are transported in turbulent plumes, which result in rapid concentration fluctuations1,2 that contain rich information about the olfactory scenery, such as the composition and location of an odour source2-4. However, it is unclear whether the mammalian olfactory system can use the underlying temporal structure to extract information about the environment. Here we show that ten-millisecond odour pulse patterns produce distinct responses in olfactory receptor neurons. In operant conditioning experiments, mice discriminated temporal correlations of rapidly fluctuating odours at frequencies of up to 40 Hz. In imaging and electrophysiological recordings, such correlation information could be readily extracted from the activity of mitral and tufted cells-the output neurons of the olfactory bulb. Furthermore, temporal correlation of odour concentrations5 reliably predicted whether odorants emerged from the same or different sources in naturalistic environments with complex airflow. Experiments in which mice were trained on such tasks and probed using synthetic correlated stimuli at different frequencies suggest that mice can use the temporal structure of odours to extract information about space. Thus, the mammalian olfactory system has access to unexpectedly fast temporal features in odour stimuli. This endows animals with the capacity to overcome key behavioural challenges such as odour source separation5, figure-ground segregation6 and odour localization7 by extracting information about space from temporal odour dynamics.

Spatial information from the odour environment in mammalian olfaction.

The sense of smell is an essential modality for many species, in particular nocturnal and crepuscular mammals, to gather information about their environment. Olfactory cues provide information over a large range of distances, allowing behaviours ranging from simple detection and recognition of objects, to tracking trails and navigating using odour plumes from afar. In this review, we discuss the features of the natural olfactory environment and provide a brief overview of how odour information can be sampled and might be represented and processed by the mammalian olfactory system. Finally, we discuss recent behavioural approaches that address how mammals extract spatial information from the environment in three different contexts: odour trail tracking, odour plume tracking and, more general, olfactory-guided navigation. Recent technological developments have seen the spatiotemporal aspect of mammalian olfaction gain significant attention, and we discuss both the promising aspects of rapidly developing paradigms and stimulus control technologies as well as their limitations. We conclude that, while still in its beginnings, research on the odour environment offers an entry point into understanding the mechanisms how mammals extract information about space.

Extracellular pH regulates excitability of vomeronasal sensory neurons.

The mouse vomeronasal organ (VNO) plays a critical role in semiochemical detection and social communication. Vomeronasal stimuli are typically secreted in various body fluids. Following direct contact with urine deposits or other secretions, a peristaltic vascular pump mediates fluid entry into the recipient's VNO. Therefore, while vomeronasal sensory neurons (VSNs) sample various stimulatory semiochemicals dissolved in the intraluminal mucus, they might also be affected by the general physicochemical properties of the "solvent." Here, we report cycle stage-correlated variations in urinary pH among female mice. Estrus-specific pH decline is observed exclusively in urine samples from sexually experienced females. Moreover, patch-clamp recordings in acute VNO slices reveal that mouse VSNs reliably detect extracellular acidosis. Acid-evoked responses share the biophysical and pharmacological hallmarks of the hyperpolarization-activated current Ih. Mechanistically, VSN acid sensitivity depends on a pH-induced shift in the voltage-dependence of Ih activation that causes the opening of HCN channels at rest, thereby increasing VSN excitability. Together, our results identify extracellular acidification as a potent activator of vomeronasal Ih and suggest HCN channel-dependent vomeronasal gain control of social chemosignaling. Our data thus reveal a potential mechanistic basis for stimulus pH detection in rodent chemosensory communication.

Co-expression of anoctamins in cilia of olfactory sensory neurons.

Vertebrates can sense and identify a vast array of chemical cues. The molecular machinery involved in chemodetection and transduction is expressed within the cilia of olfactory sensory neurons. Currently, there is only limited information available on the distribution and density of individual signaling components within the ciliary compartment. Using super-resolution microscopy, we show here that cyclic-nucleotide-gated channels and calcium-activated chloride channels of the anoctamin family are localized to discrete microdomains in the ciliary membrane. In addition to ANO2, a second anoctamin, ANO6, also localizes to ciliary microdomains. This observation, together with the fact that ANO6 and ANO2 co-localize, indicates a role for ANO6 in olfactory signaling. We show that both ANO2 and ANO6 can form heteromultimers and that this heteromerization alters the recombinant channels' physiological properties. Thus, we provide evidence for interaction of ANO2 and ANO6 in olfactory cilia, with possible physiological relevance for olfactory signaling.

Getting out the caliper: Behavioral quantification of perceptual odor similarity.

Rigorously quantifying perceptual similarity is essential to link sensory stimuli to neural activity and to define the dimensionality of perceptual space, which is challenging for the chemical senses in particular. Nakayama, Gerkin, and Rinberg present an efficient delayed match-to-sample behavioral paradigm that promises to provide a metric for odor similarity.

Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy.

Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time.

Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography and volume electron microscopy.

Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues.

jULIEs: nanostructured polytrodes for low traumatic extracellular recordings and stimulation in the mammalian brain.

Objective.Extracellular microelectrode techniques are the most widely used approach to interrogate neuronal populations. However, regardless of the manufacturing method used, damage to the vasculature and circuit function during probe insertion remains a concern. This issue can be mitigated by minimising the footprint of the probe used. Reducing the size of probes typically requires either a reduction in the number of channels present in the probe, or a reduction in the individual channel area. Both lead to less effective coupling between the probe and extracellular signals of interest.Approach.Here, we show that continuously drawn SiO2-insulated ultra-microelectrode fibres offer an attractive substrate to address these challenges. Individual fibres can be fabricated to >10 m continuous stretches and a selection of diameters below 30µm with low resistance (<100 Ω mm-1) continuously conductive metal core of <10µm and atomically flat smooth shank surfaces. To optimize the properties of the miniaturised electrode-tissue interface, we electrodeposit rough Au structures followed by ∼20 nm IrOx film resulting in the reduction of the interfacial impedance to <500 kΩ at 1 kHz.Main results. We demonstrate that these ultra-low impedance electrodes can record and stimulate both single and multi-unit activity with minimal tissue disturbance and exceptional signal-to-noise ratio in both superficial (∼40µm) and deep (∼6 mm) structures of the mouse brain. Further, we show that sensor modifications are stable and probe manufacturing is reproducible.Significance.Minimally perturbing bidirectional neural interfacing can reveal circuit function in the mammalian brainin vivo.

DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential.

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential.

Molecular characterization of projection neuron subtypes in the mouse olfactory bulb.

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.

Respiration-Locking of Olfactory Receptor and Projection Neurons in the Mouse Olfactory Bulb and Its Modulation by Brain State.

For sensory systems of the brain, the dynamics of an animal's own sampling behavior has a direct consequence on ensuing computations. This is particularly the case for mammalian olfaction, where a rhythmic flow of air over the nasal epithelium entrains activity in olfactory system neurons in a phenomenon known as sniff-locking. Parameters of sniffing can, however, change drastically with brain states. Coupled to the fact that different observation methods have different kinetics, consensus on the sniff-locking properties of neurons is lacking. To address this, we investigated the sniff-related activity of olfactory sensory neurons (OSNs), as well as the principal neurons of the olfactory bulb (OB), using 2-photon calcium imaging and intracellular whole-cell patch-clamp recordings in vivo, both in anesthetized and awake mice. Our results indicate that OSNs and OB output neurons lock robustly to the sniff rhythm, but with a slight temporal shift between behavioral states. We also observed a slight delay between methods. Further, the divergent sniff-locking by tufted cells (TCs) and mitral cells (MCs) in the absence of odor can be used to determine the cell type reliably using a simple linear classifier. Using this classification on datasets where morphological identification is unavailable, we find that MCs use a wider range of temporal shifts to encode odors than previously thought, while TCs have a constrained timing of activation due to an early-onset hyperpolarization. We conclude that the sniff rhythm serves as a fundamental rhythm but its impact on odor encoding depends on cell type, and this difference is accentuated in awake mice.

Expression patterns of chloride transporters in the auditory brainstem of developing chicken.

GABAergic transmission changes from depolarization to hyperpolarization in most vertebrate brain regions during development. By contrast, in the auditory brainstem of chicken a depolarizing effect of GABA persists after hatching. Since auditory brainstem neurons that receive GABAergic input have a Cl- reversal potential above resting membrane potential, a specifically tuned activity of Cl- transporters is likely. We here present a developmental study of the expression patterns of several members of the SLC12 family (NKCC1, NKCC2, KCC1, KCC2, KCC4, CCC6, CCC9) and of AE3 at developmental ages E7, E10, E12, E15, E17, and P1 with quantitative RT-PCR. NKCC2 and CCC9 were not detected in auditory brainstem (positive control: kidney). KCC1, CCC6 and AE3 were expressed, but not regulated, while NKCC1, KCC2 and KCC4 were regulated. The expression of the latter transporters increased, with KCC2 exhibiting the strongest expression at all time points. Biochemical analysis of the protein expression of NKCC1, KCC2 and KCC4 corroborated the findings on the mRNA level. All three transporters showed a localization at the outer rim of the cells, with NKCC1 and KCC2 expressed in neurons, and KCC4 predominantly in glia. The comparison of the published chloride reversal potential and expression of transporter proteins suggest strong differences in the efficiency of the three transporters. Further, the strong KCC2 expression could reflect a role in the structural development of auditory brainstem synapses that might lead to changes in the physiological properties.

An ancestral TMEM16 homolog from Dictyostelium discoideum forms a scramblase.

TMEM16 proteins are a recently identified protein family comprising Ca2+-activated Cl- channels that generate outwardly rectifying ionic currents in response to intracellular Ca2+ elevations. Some TMEM16 family members, such as TMEM16F/ANO6 are also essential for Ca2+-dependent phospholipid scrambling. TMEM16-like genes are present in the genomes of most eukaryotic species, the function(s) of TMEM16 family members from evolutionary ancient eukaryotes is not completely clear. Here, we provide insight into the evolution of these TMEM16 proteins by similarity searches for ancestral sequences. All eukaryotic genomes contain TMEM16 homologs, but only vertebrates have the full repertoire of ten distinct subtypes. TMEM16 homologs studied so far belong to the opisthokont branch of the phylogenetic tree, which includes the animal and fungal kingdoms. An organism outside this group is Dictyostelium discoideum, a representative of the amoebozoa group that diverged from the metazoa before fungi. We here functionally investigated the TMEM16 family member from Dictyostelium discoideum. When recombinantly expressed in HEK293 cells, DdTMEM16 induces phospholipid scrambling. However, in several electrophysiological experiments we did not find evidence for a Ca2+-activated Cl- channel function of DdTMEM16.

Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity.

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake.

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs. The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

CD36 is involved in oleic acid detection by the murine olfactory system.

Olfactory signals influence food intake in a variety of species. To maximize the chances of finding a source of calories, an animal's preference for fatty foods and triglycerides already becomes apparent during olfactory food search behavior. However, the molecular identity of both receptors and ligands mediating olfactory-dependent fatty acid recognition are, so far, undescribed. We here describe that a subset of olfactory sensory neurons expresses the fatty acid receptor CD36 and demonstrate a receptor-like localization of CD36 in olfactory cilia by STED microscopy. CD36-positive olfactory neurons share olfaction-specific transduction elements and project to numerous glomeruli in the ventral olfactory bulb. In accordance with the described roles of CD36 as fatty acid receptor or co-receptor in other sensory systems, the number of olfactory neurons responding to oleic acid, a major milk component, in Ca(2+) imaging experiments is drastically reduced in young CD36 knock-out mice. Strikingly, we also observe marked age-dependent changes in CD36 localization, which is prominently present in the ciliary compartment only during the suckling period. Our results support the involvement of CD36 in fatty acid detection by the mammalian olfactory system.

Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ.

The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology.