Reiko Kobayakawa

Innate versus learned odour processing in the mouse olfactory bulb

The mammalian olfactory system mediates various responses, including aversive behaviours to spoiled foods and fear responses to predator odours. In the olfactory bulb, each glomerulus represents a single species of odorant receptor. Because a single odorant can interact with several different receptor species, the odour information received in the olfactory epithelium is converted to a topographical map of multiple glomeruli activated in distinct areas in the olfactory bulb. To study how the odour map is interpreted in the brain, we generated mutant mice in which olfactory sensory neurons in a specific area of the olfactory epithelium are ablated by targeted expression of the diphtheria toxin gene. Here we show that, in dorsal-zone-depleted mice, the dorsal domain of the olfactory bulb was devoid of glomerular structures, although second-order neurons were present in the vacant areas. The mutant mice lacked innate responses to aversive odorants, even though they were capable of detecting them and could be conditioned for aversion with the remaining glomeruli. These results indicate that, in mice, aversive information is received in the olfactory bulb by separate sets of glomeruli, those dedicated for innate and those for learned responses.

The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor

Various social behaviours in mice are regulated by chemical signals called pheromones that act through the vomeronasal system. Exocrine gland-secreting peptide 1 (ESP1) is a 7-kDa peptide that is released into male tear fluids and stimulates vomeronasal sensory neurons in female mice. Here, we describe the molecular and neural mechanisms that are involved in the decoding of ESP1 signals in the vomeronasal system, which leads to behavioural output in female mice. ESP1 is recognized by a specific vomeronasal receptor, V2Rp5, and the ligand–receptor interaction results in sex-specific signal transmission to the amygdaloid and hypothalamic nuclei via the accessory olfactory bulb. Consequently, ESP1 enhances female sexual receptive behaviour upon male mounting (lordosis), allowing successful copulation. In V2Rp5-deficient mice, ESP1 induces neither neural activation nor sexual behaviour. These findings show that ESP1 is a crucial male pheromone that regulates female reproductive behaviour through a specific receptor in the mouse vomeronasal system.

Pre-Target Axon Sorting Establishes the Neural Map Topography

Mapping the Neuronal Map In vertebrates, sensory information is topographically represented as a neural map in the brain. How is the neural map formed in the brain? Nearly a half-century ago, Roger Sperry proposed the “chemoaffinity” model, in which the positional cues on the target determine the axonal projection site, thereby establishing the topographic neural map. However, molecular mechanisms of topographic map formation remain controversial. Imai et al. (p. 585, published online 9 July; see the Perspective by Miyamichi and Luo) now report that the topographic map is formed by axon-axon interactions before the axons reach the target. In the mouse olfactory system, the topography of the map is determined by the relative expression levels of a guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, expressed in axons. Topographic organization occurs even in the absence of the target, the olfactory bulb. These findings require that Sperrys model, which suggests that only the targets determine the topography of neural maps, needs to be reconsidered. The mouse olfactory topographic neural map is self-organized by interactions between axons, not directed by the target. Sensory information detected by the peripheral nervous system is represented as a topographic map in the brain. It has long been thought that the topography of the map is determined by graded positional cues that are expressed by the target. Here, we analyzed the pre-target axon sorting for olfactory map formation in mice. In olfactory sensory neurons, an axon guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, are expressed in a complementary manner. We found that expression levels of Neuropilin-1 determined both pre-target sorting and projection sites of axons. Olfactory sensory neuron–specific knockout of Semaphorin-3A perturbed axon sorting and altered the olfactory map topography. Thus, pre-target axon sorting plays an important role in establishing the topographic order based on the relative levels of guidance molecules expressed by axons.

Parallel Mitral and Tufted Cell Pathways Route Distinct Odor Information to Different Targets in the Olfactory Cortex

Odor signals are conveyed from the olfactory bulb to the olfactory cortex (OC) by mitral cells (MCs) and tufted cells (TCs). However, whether and how the two types of projection neuron differ in function and axonal connectivity is still poorly understood. Odor responses and axonal projection patterns were compared between MCs and TCs in mice by visualizing axons of electrophysiologically identified single neurons. TCs demonstrated shorter onset latency for reliable responses than MCs. The shorter latency response of TCs was maintained in a wide range of odor concentrations, whereas MCs responded only to strong signals. Furthermore, individual TCs projected densely to focal targets only in anterior areas of the OC, whereas individual MCs dispersedly projected to all OC areas. Surprisingly, in anterior OC areas, the two cell types projected to segregated subareas. These results suggest that MCs and TCs transmit temporally distinct odor information to different OC targets.

Elimination of Adult-Born Neurons in the Olfactory Bulb Is Promoted during the Postprandial Period

Granule cells (GCs) in the mouse olfactory bulb (OB) continue to be generated in adulthood, with nearly half incorporated and the remainder eliminated. Here, we show that elimination of adult-born GCs is promoted during a short time window in the postprandial period. Under restricted feeding, the number of apoptotic GCs specifically increased within a few hours after the start of feeding. This enhanced GC apoptosis occurred in association with postprandial behaviors that included grooming, resting, and sleeping, and was particularly correlated with the length of postprandial sleep. Further, deprivation of olfactory sensory experience in the local OB area potentiated the extent of GC elimination in that area during the postprandial period. Sensory experience-dependent enhancement of GC elimination also occurred during postprandial period under natural feeding condition. These results suggest that extensive structural reorganization of bulbar circuitry occurs during the postprandial period, reflecting sensory experience during preceding waking period.

Spatial Arrangement of Glomerular Molecular-Feature Clusters in the Odorant-Receptor Class Domains of the Mouse Olfactory Bulb

The glomerular layer of the mammalian olfactory bulb (OB) forms odorant receptor (OR) maps. Each OR map is structurally and functionally compartmentalized into zones (dorsal and ventral) and domains (DI and DII in the dorsal zone). We previously reported that glomeruli with similar molecular receptive range properties formed molecular feature clusters at stereotypical positions in the rat OB. However, the spatial arrangement of the molecular feature clusters with regard to the OR zones and domains has not been systematically examined. In this study, we optically mapped the molecular feature clusters of glomeruli within the domain and zone framework of the OB using domain-visible class II GFP transgenic mice. In all mice examined, fatty acid-responsive cluster A was located in the lateral part of domain DI, whereas clusters B, C, and D were arranged in an anterior to posterior order within domain DII. We also found a new cluster of glomeruli that respond to fox odor trimethyl-thiazoline and its structural analogs (heterocyclic odorants that contain sulfur and nitrogen atoms within the ring). This cluster (named cluster J) was located posterior to cluster D within the DII domain. These results show that molecular feature clusters correspond to specific subsets of glomeruli in selective domains of the OR map, suggesting that the molecular feature clusters represent specific ORs that have similar molecular receptive range properties and functional roles.

Genetic dissection of pheromone processing reveals main olfactory system-mediated social behaviors in mice

Significance It is now widely accepted that the range of pheromones that control social behaviors are processed by both the vomeronasal system (VNS) and the main olfactory system (MOS). However, the functional contributions of each subsystem in social behavior remain unclear. Here, we showed that mice with loss-of-function confined to the dorsal MOS maintained innate odor recognition and VNS activity, but failed to demonstrate multiple male and female social behaviors. Functional dissociation of the MOS and VNS enabled the identification of an MOS-mediated processing of semiochemical information, independent of the VNS. Most mammals have two major olfactory subsystems: the main olfactory system (MOS) and vomeronasal system (VNS). It is now widely accepted that the range of pheromones that control social behaviors are processed by both the VNS and the MOS. However, the functional contributions of each subsystem in social behavior remain unclear. To genetically dissociate the MOS and VNS functions, we established two conditional knockout mouse lines that led to either loss-of-function in the entire MOS or in the dorsal MOS. Mice with whole-MOS loss-of-function displayed severe defects in active sniffing and poor survival through the neonatal period. In contrast, when loss-of-function was confined to the dorsal MOB, sniffing behavior, pheromone recognition, and VNS activity were maintained. However, defects in a wide spectrum of social behaviors were observed: attraction to female urine and the accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and risk-assessment behaviors in response to an alarm pheromone. Functional dissociation of pheromone detection and pheromonal induction of behaviors showed the anterior olfactory nucleus (AON)-regulated social behaviors downstream from the MOS. Lesion analysis and neural activation mapping showed pheromonal activation in multiple amygdaloid and hypothalamic nuclei, important regions for the expression of social behavior, was dependent on MOS and AON functions. Identification of the MOS-AON–mediated pheromone pathway may provide insights into pheromone signaling in animals that do not possess a functional VNS, including humans.

Htr2a-Expressing Cells in the Central Amygdala Control the Hierarchy between Innate and Learned Fear

Fear is induced by innate and learned mechanisms involving separate pathways. Here, we used an olfactory-mediated innate-fear versus learned-fear paradigm to investigate how these pathways are integrated. Notably, prior presentation of innate-fear stimuli inhibited learned-freezing response, but not vice versa. Whole-brain mapping and pharmacological screening indicated that serotonin-2A receptor (Htr2a)-expressing cells in the central amygdala (CeA) control both innate and learned freezing, but in opposing directions. In vivo fiber photometry analyses in freely moving mice indicated that innate but not learned-fear stimuli suppressed the activity of Htr2a-expressing CeA cells. Artificial inactivation of these cells upregulated innate-freezing response and downregulated learned-freezing response. Thus, Htr2a-expressing CeA cells serve as a hierarchy generator, prioritizing innate fear over learned fear.

Supersensitive detection and discrimination of enantiomers by dorsal olfactory receptors: evidence for hierarchical odour coding

Enantiomeric pairs of mirror-image molecular structures are difficult to resolve by instrumental analyses. The human olfactory system, however, discriminates (−)-wine lactone from its (+)-form rapidly within seconds. To gain insight into receptor coding of enantiomers, we compared behavioural detection and discrimination thresholds of wild-type mice with those of ΔD mice in which all dorsal olfactory receptors are genetically ablated. Surprisingly, wild-type mice displayed an exquisite “supersensitivity” to enantiomeric pairs of wine lactones and carvones. They were capable of supersensitive discrimination of enantiomers, consistent with their high detection sensitivity. In contrast, ΔD mice showed selective major loss of sensitivity to the (+)-enantiomers. The resulting 108-fold differential sensitivity of ΔD mice to (−)- vs. (+)-wine lactone matched that observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)-enantiomer, similarly to ΔD mice. Moreover, ΔD mice showed >1010-fold reductions in enantiomer discrimination sensitivity compared to wild-type mice. ΔD mice detected one or both of the (−)- and (+)-enantiomers over a wide concentration range, but were unable to discriminate them. This “enantiomer odour discrimination paradox” indicates that the most sensitive dorsal receptors play a critical role in hierarchical odour coding for enantiomer identification.

Regulation of survival and death of adult-born neurons in the local area of the olfactory bulb by local sensory input

Subventricular zone (SVZ) of adult brain has neural stem/progenitor cells (NPCs) that generate new neurons throughout life. In the NPCs, intracellular Ca2+ is known to play a critical role in regulating different stages of early brain development and neurogenesis. To elucidate participation of Ca2+ signaling pathway in proliferation of the NPCs, we evaluated the effect of ryanodine receptor (RyR) inhibitor dantrolene on proliferative activity in the NPCs derived from the SVZ of adult mice. Cells were prepared from the SVZ of 5-week-old Std-ddY male mice and then primarily cultured in DMEM/F12 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 8 days in vitro (DIV). After replating, the cells were secondarily cultured for 5 DIV under the same conditions in the absence or presence of dantrolene for a period of 8–13 DIV. In the presence of both EGF and bFGF, marked round spheres were formed, continued to grow, and proliferate to form large neurospheres. In addition, most cells obtained from the neurospheres were immunoreactive to nestin. To determine the expression of RyR subunits in the NPCs, we performed RT-PCR analysis using total RNA prepared from the NPCs. Although there exist 3 subunits of RyR in the NPCs, RyR3 was the highest level of 3 subunits of RyR. ELISA for 5′-bromo2′-deoxyuridine revealed that a marked decrease in the proliferative activity was seen by treatment with dantrolene in a dose-dependent manner (2, 10, 20 or 50 M). However, dantrolene was ineffective in releasing lactate dehydrogenase into the culture medium. Moreover, treatment with dantrolene resulted in a marked reduction in STAT3 and Pax6 mRNA in NPCs. These results suggest that Ca2+ release from the endoplasmic reticulum via the RyR would positively regulate proliferative activity in NPCs of adult mouse SVZ.