Shin Nagayama

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.

In Vivo Simultaneous Tracing and Ca2+ Imaging of Local Neuronal Circuits

A central question about the brain is how information is processed by large populations of neurons embedded in intricate local networks. Answering this question requires not only monitoring functional dynamics of many neurons simultaneously, but also interpreting such activity patterns in the context of neuronal circuitry. Here, we introduce a versatile approach for loading Ca(2+) indicators in vivo by local electroporation. With this method, Ca(2+) imaging can be performed both at neuron population level and with exquisite subcellular resolution down to dendritic spines and axon boutons. This enabled mitral cell odor-evoked ensemble activity to be analyzed simultaneously with revealing their specific connectivity to different glomeruli. Colabeling of Purkinje cell dendrites and intersecting parallel fibers allowed Ca(2+) imaging of both presynaptic boutons and postsynaptic dendrites. This approach thus provides an unprecedented capability for in vivo visualizing active cell ensembles and tracing their underlying local neuronal circuits.

Differential axonal projection of mitral and tufted cells in the mouse main olfactory system

In the past decade, much has been elucidated regarding the functional organization of the axonal connection of olfactory sensory neurons to olfactory bulb (OB) glomeruli. However, the manner in which projection neurons of the OB process odorant input and send this information to higher brain centers remains unclear. Here, we report long-range, large-scale tracing of the axonal projection patterns of OB neurons using two-photon microscopy. Tracer injection into a single glomerulus demonstrated widely distributed mitral/tufted cell axonal projections on the lateroventral surface of the mouse brain, including the anterior/posterior piriform cortex (PC) and olfactory tubercle (OT). We noted two distinct groups of labeled axons: PC-orienting axons and OT-orienting axons. Each group occupied distinct parts of the lateral olfactory tract. PC-orienting axons projected axon collaterals to a wide area of the PC but only a few collaterals to the OT. OT-orienting axons densely projected axon collaterals primarily to the anterolateral OT (alOT). Different colored dye injections into the superficial and deep portions of the OB external plexiform layer revealed that the PC-orienting axon populations originated in presumed mitral cells and the OT-orienting axons in presumed tufted cells. These data suggest that although mitral and tufted cells receive similar odor signals from a shared glomerulus, they process the odor information in different ways and send their output to different higher brain centers via the PC and alOT.

Neuronal organization of olfactory bulb circuits

Olfactory sensory neurons extend their axons solely to the olfactory bulb, which is dedicated to odor information processing. The olfactory bulb is divided into multiple layers, with different types of neurons found in each of the layers. Therefore, neurons in the olfactory bulb have conventionally been categorized based on the layers in which their cell bodies are found; namely, juxtaglomerular cells in the glomerular layer, tufted cells in the external plexiform layer, mitral cells in the mitral cell layer, and granule cells in the granule cell layer. More recently, numerous studies have revealed the heterogeneous nature of each of these cell types, allowing them to be further divided into subclasses based on differences in morphological, molecular, and electrophysiological properties. In addition, technical developments and advances have resulted in an increasing number of studies regarding cell types other than the conventionally categorized ones described above, including short-axon cells and adult-generated interneurons. Thus, the expanding diversity of cells in the olfactory bulb is now being acknowledged. However, our current understanding of olfactory bulb neuronal circuits is mostly based on the conventional and simplest classification of cell types. Few studies have taken neuronal diversity into account for understanding the function of the neuronal circuits in this region of the brain. This oversight may contribute to the roadblocks in developing more precise and accurate models of olfactory neuronal networks. The purpose of this review is therefore to discuss the expanse of existing work on neuronal diversity in the olfactory bulb up to this point, so as to provide an overall picture of the olfactory bulb circuit.

Optical Imaging of Postsynaptic Odor Representation in the Glomerular Layer of the Mouse Olfactory Bulb

Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca(2+)-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca(2+) indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.

Detection and Masking of Spoiled Food Smells by Odor Maps in the Olfactory Bulb

Two major causes of spoiled food smells such as fatty, fishy off-flavors are alkylamines liberated by bacterial actions and aliphatic acids-aldehydes generated by lipid oxidation. Using the method of intrinsic signal imaging, we mapped alkylamine-responsive glomeruli to a subregion of the aliphatic acid-responsive and aldehyde-responsive cluster in the odor maps of rat olfactory bulb. Extracellular single-unit recordings from mitral-tufted cells in the subregion showed that individual cells responded to the alkylamines in addition to acids and aldehydes. Responses of mitral-tufted cells tended to last for a long period (5-88 sec) even after the cessation of the alkylamine stimulation. These results suggest that the subregion is part of the representation of the fatty, fishy odor quality. Fennel and clove, spices known to add flavor and mask the fatty, fishy odor, activated glomeruli in the surrounding clusters and suppressed the alkylamine-induced and acid-aldehyde-induced responses of mitral cells, suggesting that the odor masking is mediated, in part, by lateral inhibitory connections in the odor maps of the olfactory bulb.

Molecular‐feature domains with posterodorsal–anteroventral polarity in the symmetrical sensory maps of the mouse olfactory bulb: mapping of odourant‐induced Zif268 expression

Individual glomeruli in the mammalian olfactory bulb presumably represent a single type of odourant receptor. Thus, the glomerular sheet provides odourant receptor maps at the surface of the olfactory bulb. To understand the basic spatial organization of the olfactory sensory maps, we first compared the spatial distribution of odourant‐induced responses measured by the optical imaging of intrinsic signals with that detected immunohistochemically by expressions of Zif268, one of the immediate early gene products in juxtaglomerular cells. In the dorsal surface of the bulb, we detected a clear correlation in the spatial pattern between these responses. In addition, the molecular‐feature domains and their polarities (spatial shifts of responses with an increase in carbon chain length) that were defined by the optical imaging method could be also detected by the Zif268 mapping method. We then mapped the Zif268 signals over the entire olfactory bulb using a homologous series of fatty acids and aliphatic alcohols as stimulus odourants. We superimposed the Zif268 signals onto the standard unrolled map with the help of cell adhesion molecule compartments. Each odourant typically elicited two pairs of clusters of dense Zif268 signals. The results showed that molecular‐feature domains and their polarities were arranged symmetrically at stereotypical positions in a mirror‐image fashion between the lateral and the medial sensory maps. The polarity of each domain was roughly in parallel with the posterodorsal–anteroventral axis that was defined by the cell adhesion molecule compartments. These results suggest that the molecular‐feature domain with its fixed polarity is one of the basic structural units in the spatial organization of the odourant receptor maps in the olfactory bulb.

Sensory Deprivation Disrupts Homeostatic Regeneration of Newly Generated Olfactory Sensory Neurons after Injury in Adult Mice

Although it is well known that injury induces the generation of a substantial number of new olfactory sensory neurons (OSNs) in the adult olfactory epithelium (OE), it is not well understood whether olfactory sensory input influences the survival and maturation of these injury-induced OSNs in adults. Here, we investigated whether olfactory sensory deprivation affected the dynamic incorporation of newly generated OSNs 3, 7, 14, and 28 d after injury in adult mice. Mice were unilaterally deprived of olfactory sensory input by inserting a silicone tube into their nostrils. Methimazole, an olfactotoxic drug, was also injected intraperitoneally to bilaterally ablate OSNs. The OE was restored to its preinjury condition with new OSNs by day 28. No significant differences in the numbers of olfactory marker protein-positive mature OSNs or apoptotic OSNs were observed between the deprived and nondeprived sides 0–7 d after injury. However, between days 7 and 28, the sensory-deprived side showed markedly fewer OSNs and mature OSNs, but more apoptotic OSNs, than the nondeprived side. Intrinsic functional imaging of the dorsal surface of the olfactory bulb at day 28 revealed that responses to odor stimulation were weaker in the deprived side compared with those in the nondeprived side. Furthermore, prevention of cell death in new neurons 7–14 d after injury promoted the recovery of the OE. These results indicate that, in the adult OE, sensory deprivation disrupts compensatory OSN regeneration after injury and that newly generated OSNs have a critical time window for sensory-input-dependent survival 7–14 d after injury.

Parallel tufted cell and mitral cell pathways from the olfactory bulb to the olfactory cortex

In the mammalian olfactory system, sniff-induced odor signals are conveyed from the olfactory bulb to the olfactory cortex by two types of projection neurons, tufted cells and mitral cells. This chapter summarizes recent advances in knowledge of the structural and functional differences between tufted cell and mitral cell circuits. Tufted cells and mitral cells show distinct patterns of lateral dendrite projection and make dendrodendritic reciprocal synaptic connections with different subtypes of granule cell inhibitory interneurons. Tufted cells and mitral cells thus form distinct local circuits within the olfactory bulb: small-scale tufted cell dendrodendritic circuits and larger-scale mitral cell dendrodendritic circuits. In addition, tufted cells and mitral cells differ dramatically in their axonal projection to the olfactory cortex. Individual tufted cells project axons to focal targets in the olfactory peduncle areas, whereas individual mitral cells send axons in a dispersed way to nearly all areas of the olfactory cortex, including nearly all parts of the piriform cortex. Furthermore, tufted cells and mitral cells differ strikingly in how they respond to odor inhalation. Compared with mitral cells, tufted cells show earlier-onset, higher-frequency spike discharges. Tufted cells are activated at a much lower odor concentration threshold than activating mitral cells. During an inhalation–exhalation sniff cycle, tufted cell circuits generate early-onset fast gamma oscillation while mitral cell circuits give rise to later-onset slow gamma oscillation. From these structural and functional differences, we hypothesize that the two types of projection neurons play distinct roles in sending sniff-induced odor signals to the olfactory cortex. Specifically, tufted cells provide specificity-projecting circuits that send specific odor information to focal targets in the olfactory peduncle areas with early-onset fast gamma synchronization. In contrast, mitral cells give rise to dispersed-projection feed-forward “binding” circuits that transmit the response synchronization timing via their later-onset slow gamma synchronization to pyramidal cells distributed across all parts of the piriform cortex.

In Vivo Local Dye Electroporation for Ca2+ Imaging and Neuronal-Circuit Tracing

A major technical challenge for using optical imaging to analyze neuronal circuit functions is how to effectively load synthetic Ca(2+) dyes or neural tracers into the brain. We introduce here a simple but versatile approach to label many neurons and clearly visualize their axonal and dendritic morphology. The method uses a large-tip patch pipette filled with dextran-conjugated Ca(2+) dyes or fluorescent tracers. By inserting the pipette into a targeted brain area and passing microampere current pulses, dyes or tracers are electroporated into dendrites and axons near the pipette tip. The dyes are then transported to reveal the entire cell morphology, suitable for both functional Ca(2+) imaging and neuronal circuit tracing. This process requires basic physiological equipment normally available in a physiological laboratory.