Olfactory Circuitry and Behavioral Decisions.

In mammals, odor information detected by olfactory sensory neurons is converted to a topographic map of activated glomeruli in the olfactory bulb. Mitral cells and tufted cells transmit signals sequentially to the olfactory cortex for behavioral outputs. To elicit innate behavioral responses, odor signals are directly transmitted by distinct subsets of mitral cells from particular functional domains in the olfactory bulb to specific amygdala nuclei. As for the learned decisions, input signals are conveyed by tufted cells as well as by mitral cells to the olfactory cortex. Behavioral scene cells link the odor information to the valence cells in the amygdala to elicit memory-based behavioral responses. Olfactory decision and perception take place in relation to the respiratory cycle. How is the sensory quality imposed on the olfactory inputs for behavioral outputs? How are the two types of odor signals, innate and learned, processed during respiration? Here, we review recent progress on the study of neural circuits involved in decision making in the mouse olfactory system.

TRIM67 Implicates in Regulating the Homeostasis and Synaptic Development of Mitral Cells in the Olfactory Bulb.

In recent years, olfactory dysfunction has attracted increasingly more attention as a hallmark symptom of neurodegenerative diseases (ND). Deeply understanding the molecular basis underlying the development of the olfactory bulb (OB) will provide important insights for ND studies and treatments. Now, with a genetic knockout mouse model, we show that TRIM67, a new member of the tripartite motif (TRIM) protein family, plays an important role in regulating the proliferation and development of mitral cells in the OB. TRIM67 is abundantly expressed in the mitral cell layer of the OB. The genetic deletion of TRIM67 in mice leads to excessive proliferation of mitral cells in the OB and defects in its synaptic development, resulting in reduced olfactory function in mice. Finally, we show that TRIM67 may achieve its effect on mitral cells by regulating the Semaphorin 7A/Plexin C1 (Sema7A/PlxnC1) signaling pathway.

Neurotransmitter regulation rather than cell-intrinsic properties shapes the high-pass filtering properties of olfactory bulb glomeruli.

GABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate an intraglomerular 'high-pass' filter through inhibition targeted onto a glomerulus. With this mechanism, weak stimuli (e.g. an odour with a low affinity for an odourant receptor) mainly produce PG cell-driven inhibition but strong stimuli generate enough excitation to overcome inhibition. PG cells may be particularly susceptible to being activated by weak stimuli due to their intrinsically small size and high input resistance. Here, we used dual-cell patch-clamp recordings and imaging methods in bulb slices obtained from wild-type and transgenic rats with labelled GABAergic cells to test a number of predictions of the high-pass filtering model. We first directly compared the responsiveness of PG cells with that of external tufted cells (eTCs), which are a class of excitatory cells that reside in a parallel but opposing position in the glomerular circuitry. PG cells were in fact found to be no more responsive than eTCs at low levels of sensory neuron activity. While PG cells required smaller currents to be excited, this advantage was offset by the fact that a given level of sensory neuron activity produced much smaller synaptic currents. We did, however, identify other factors that shaped the excitation/inhibition balance in a manner that would support a high-pass filter, including glial glutamate transporters and presynaptic metabotropic glutamate receptors. We conclude that a single glomerulus may act as a high-pass filter to enhance the contrast between different olfactory stimuli through mechanisms that are largely independent cell-intrinsic properties. KEY POINTS: GABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate a 'high-pass' filter at a single glomerulus that selectively blocks weak stimulus signals. Their efficacy may reflect their intrinsically small size and high input resistance, which allows them to be easily excited. It was found that PG cells were in fact no more likely to be activated by weak stimuli than excitatory neurons. PG cells fired action potentials more readily in response to a fixed current input, but this advantage for excitability was offset by small synaptic currents. Glomeruli nevertheless display an excitation/inhibition balance that can support a high-pass filter, shifting from unfavourable to favourable with increasing stimulus strength. Factors shaping the filter include glial glutamate transporters and presynaptic metabotropic glutamate receptors. It is concluded that a single glomerulus may act as a high-pass filter to enhance stimulus contrast through mechanisms that are largely independent of cell-intrinsic properties.

Synchronous Infra-Slow Oscillations Organize Ensembles of Accessory Olfactory Bulb Projection Neurons into Distinct Microcircuits.

The accessory olfactory system controls social and sexual behavior. In the mouse accessory olfactory bulb, the first central stage of information processing along the accessory olfactory pathway, projection neurons (mitral cells) display infra-slow oscillatory discharge with remarkable periodicity. The physiological mechanisms that underlie this default output state, however, remain controversial. Moreover, whether such rhythmic infra-slow activity patterns exist in awake behaving mice and whether such activity reflects the functional organization of the accessory olfactory bulb circuitry remain unclear. Here, we hypothesize that mitral cell ensembles form synchronized microcircuits that subdivide the accessory olfactory bulb into segregated functional clusters. We use a miniature microscope to image the Ca2+ dynamics within the apical dendritic compartments of large mitral cell ensembles in vivo We show that infra-slow periodic patterns of concerted neural activity, indeed, reflect the idle state of accessory olfactory bulb output in awake male and female mice. Ca2+ activity profiles are distinct and glomerulus-specific. Confocal time-lapse imaging in acute slices reveals that groups of mitral cells assemble into microcircuits that exhibit correlated Ca2+ signals. Moreover, electrophysiological profiling of synaptic connectivity indicates functional coupling between mitral cells. Our results suggest that both intrinsically rhythmogenic neurons and neurons entrained by fast synaptic drive are key elements in organizing the accessory olfactory bulb into functional microcircuits, each characterized by a distinct default pattern of infra-slow rhythmicity.SIGNIFICANCE STATEMENT Information processing in the accessory olfactory bulb (AOB) plays a central role in conspecific chemosensory communication. Surprisingly, many basic physiological principles that underlie neuronal signaling in the AOB remain elusive. Here, we show that AOB projection neurons (mitral cells) form parallel synchronized ensembles both in vitro and in vivo Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway.

Piezo2-peripheral baroreceptor channel expressed in select neurons of the mouse brain: a putative mechanism for synchronizing neural networks by transducing intracranial pressure pulses.

Here we use immunohistochemistry to examine the expression of Piezo2 in neurons of the mouse dorsal root ganglia and brain. Whereas Piezo2 is expressed in the large majority (≥ 90%) of dorsal root ganglia neurons, Piezo2 expression is restricted to select neuron types in specific brain regions, including neocortical and hippocampal pyramidal neurons, cerebellar Purkinje cells and mitral cells of the olfactory bulb. Given the well-established role of Piezo2 as a low-threshold pressure sensor (i.e., ≤5 mmHg) in peripheral mechanosensation, including the regulation of breathing and blood pressure, its expression in central neurons has interesting implications. In particular, we hypothesize that Piezo2 provides neurons with an intrinsic resonance that promotes their entrainment by the normal intracranial pressure pulses (~5 mmHg) associated with breathing and cardiac cycles. The pressure-induced change in neural activity need only be very subtle to increase, for example, the robustness of respiration-entrained oscillations reported previously in widely distributed neuronal networks in both rodent and human brains. This idea of a "global brain rhythm" first arose from the effect of nasal airflow in activating mechanosensitive olfactory sensory neurons, which then synaptically entrain mitral cells within the olfactory bulb and through their projections, neural networks in other brain regions, including the hippocampus and neocortex. Our proposed, non-synaptic, intrinsic mechanism, where Piezo2 tracks the highly predictable and "metronome-like" intracranial pressure pulses-to date generally considered epiphenomena-would have the advantage that a physical force rapidly transmitted throughout the brain also contributes to this synchronization.

Bursting mitral cells time the oscillatory coupling between olfactory bulb and entorhinal networks in neonatal mice.

KEY POINTS: During early postnatal development, mitral cells show either irregular bursting or non-bursting firing patterns Bursting mitral cells preferentially fire during theta bursts in the neonatal olfactory bulb, being locked to the theta phase Bursting mitral cells preferentially fire during theta bursts in the neonatal lateral entorhinal cortex and are temporally related to both respiration rhythm- and theta phase Bursting mitral cells act as a cellular substrate of the olfactory drive that promotes the oscillatory entrainment of entorhinal networks ABSTRACT: Shortly after birth, the olfactory system provides not only the main source of environmental inputs to blind, deaf, non-whisking and motorically-limited rodents, but also the drive boosting the functional entrainment of limbic circuits. However, the cellular substrate of this early communication remains largely unknown. Here, we combine in vivo and in vitro patch-clamp and extracellular recordings to reveal the contribution of mitral cell (MC) firing to early patterns of network activity in both the neonatal olfactory bulb (OB) and the lateral entorhinal cortex (LEC), the gatekeeper of limbic circuits. We show that MCs predominantly fire either in an irregular bursting or non-bursting pattern during discontinuous theta events in the OB. However, the temporal spike-theta phase coupling is stronger for bursting than non-bursting MCs. In line with the direct OB-to-LEC projections, both bursting and non-bursting discharge augments during co-ordinated patterns of entorhinal activity, albeit with higher magnitude for bursting MCs. For these neurons, temporal coupling to the discontinuous theta events in the LEC is stronger. Thus, bursting MCs might drive the entrainment of the OB-LEC network during neonatal development.

Short-term plasticity in glomerular inhibitory circuits shapes olfactory bulb output.

Short-term plasticity is a fundamental synaptic property thought to underlie memory and neural processing. The glomerular microcircuit comprises complex excitatory and inhibitory interactions and transmits olfactory nerve signals to the excitatory output neurons, mitral/tufted cells (M/TCs). The major glomerular inhibitory interneurons, short axon cells (SACs) and periglomerular cells (PGCs), both provide feedforward and feedback inhibition to M/TCs and have reciprocal inhibitory synapses between each other. Olfactory input is episodically driven by sniffing. We hypothesized that frequency-dependent short-term plasticity within these inhibitory circuits could influence signals sent to higher-order olfactory networks. To assess short-term plasticity in glomerular circuits and MC outputs, we virally delivered channelrhodopsin-2 (ChR2) in glutamic acid decarboxylase-65 promotor (GAD2-cre) or tyrosine hydroxylase promoter (TH-cre) mice and selectively activated one of these two populations while recording from cells of the other population or from MCs. Selective activation of TH-ChR2-expressing SACs inhibited all recorded GAD2-green fluorescent protein(GFP)-expressing presumptive PGC cells, and activation of GAD2-ChR2 cells inhibited TH-GFP-expressing SACs, indicating reciprocal inhibitory connections. SAC synaptic inhibition of GAD2-expressing cells was significantly facilitated at 5-10 Hz activation frequencies. In contrast, GAD2-ChR2 cell inhibition of TH-expressing cells was activation-frequency independent. Both SAC and PGC inhibition of MCs also exhibited short-term plasticity, pronounced in the 5-20 Hz range corresponding to investigative sniffing frequency ranges. In paired SAC and olfactory nerve electrical stimulations, the SAC to MC synapse was able to markedly suppress MC spiking. These data suggest that short-term plasticity across investigative sniffing ranges may differentially regulate intra- and interglomerular inhibitory circuits to dynamically shape glomerular output signals to downstream targets.NEW & NOTEWORTHY Short-term plasticity is a fundamental synaptic property that modulates synaptic strength based on preceding activity of the synapse. In rodent olfaction, sensory input arrives episodically driven by sniffing rates ranging from quiescent respiration (1-2 Hz) through to investigative sniffing (5-10 Hz). Here we show that glomerular inhibitory networks are exquisitely sensitive to input frequencies and exhibit plasticity proportional to investigative sniffing frequencies. This indicates that olfactory glomerular circuits are dynamically modulated by episodic sniffing input.

Tag1 deficiency results in olfactory dysfunction through impaired migration of mitral cells.

The olfactory system provides mammals with the abilities to investigate, communicate and interact with their environment. These functions are achieved through a finely organized circuit starting from the nasal cavity, passing through the olfactory bulb and ending in various cortical areas. We show that the absence of transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice results in a significant and selective defect in the number of the main projection neurons in the olfactory bulb, namely the mitral cells. A subpopulation of these projection neurons is reduced in Tag1-deficient mice as a result of impaired migration. We demonstrate that the detected alterations in the number of mitral cells are well correlated with diminished odor discrimination ability and social long-term memory formation. Reduced neuronal activation in the olfactory bulb and the corresponding olfactory cortex suggest that Tag1 is crucial for the olfactory circuit formation in mice. Our results underpin the significance of a numerical defect in the mitral cell layer in the processing and integration of odorant information and subsequently in animal behavior.

Synaptic clusters function as odor operators in the olfactory bulb.

How the olfactory bulb organizes and processes odor inputs through fundamental operations of its microcircuits is largely unknown. To gain new insight we focus on odor-activated synaptic clusters related to individual glomeruli, which we call glomerular units. Using a 3D model of mitral and granule cell interactions supported by experimental findings, combined with a matrix-based representation of glomerular operations, we identify the mechanisms for forming one or more glomerular units in response to a given odor, how and to what extent the glomerular units interfere or interact with each other during learning, their computational role within the olfactory bulb microcircuit, and how their actions can be formalized into a theoretical framework in which the olfactory bulb can be considered to contain "odor operators" unique to each individual. The results provide new and specific theoretical and experimentally testable predictions.

Early Odorant Exposure Increases the Number of Mitral and Tufted Cells Associated with a Single Glomerulus.

The highly specific organization of the olfactory bulb (OB) is well known, but the impact of early odorant experience on its circuit structure is unclear. Olfactory sensory neurons (OSNs) project axons from the olfactory epithelium to the OB, where they form spherical neuropil structures called glomeruli. These glomeruli and the postsynaptic targets of OSNs, including mitral and tufted cells (M/TCs) and juxtaglomerular cells, form glomerular modules, which represent the basic odor-coding units of the OB. Here, we labeled M/TCs within a single glomerular module of the mouse OB and show that odorant exposure that starts prenatally and continues through postnatal day 25 has a major impact on the structure of the glomerular module. We confirm that exposure increases the volume of the activated glomeruli and show that exposure increases M/TC number by >40% in a glomerulus-specific fashion. Given the role of M/TCs in OB output and in lateral inhibition, increasing the number of M/TCs connected to a single glomerulus may also increase the influence of that glomerulus on the OB network and on OB output. Our results show that early odorant exposure has a profound effect on OB connectivity and thus may affect odorant processing significantly. SIGNIFICANCE STATEMENT: Experience shapes neural circuits in a variety of ways, most commonly by changing the strength of activated connections. Relatively little is known about how experience changes circuitry in the olfactory system. Here, we show that for a genetically identified glomerulus in the mouse olfactory bulb, early odorant exposure increases the number of associated mitral and tufted cells by 40% and 100%, respectively. Understanding the structural changes induced by early odorant experience can provide insight into how bulbar organization gives rise to efficient processing. We find that odorant experience increases the number of projection neurons associated with a single glomerulus significantly, a dramatic and long-lasting structural change that may have important functional implications.

Astroglial Connexin 43 Hemichannels Modulate Olfactory Bulb Slow Oscillations.

An emergent concept in neurosciences consists in considering brain functions as the product of dynamic interactions between neurons and glial cells, particularly astrocytes. Although the role played by astrocytes in synaptic transmission and plasticity is now largely documented, their contribution to neuronal network activity is only beginning to be appreciated. In mouse olfactory bulb slices, we observed that the membrane potential of mitral cells oscillates between UP and DOWN states at a low frequency (<1 Hz). Such slow oscillations are correlated with glomerular local field potentials, indicating spontaneous local network activity. Using a combination of genetic and pharmacological tools, we showed that the activity of astroglial connexin 43 hemichannels, opened in an activity-dependent manner, increases UP state amplitude and impacts mitral cell firing rate. This effect requires functional adenosine A1 receptors, in line with the observation that ATP is released via connexin 43 hemichannels. These results highlight a new mechanism of neuroglial interaction in the olfactory bulb, where astrocyte connexin hemichannels are both targets and modulators of neuronal circuit function. SIGNIFICANCE STATEMENT: An emergent concept in neuroscience consists in considering brain function as the product of dynamic interactions between neurons and glial cells, particularly astrocytes. A typical feature of astrocytes is their high expression level of connexins, the molecular constituents of gap junction channels and hemichannels. Although hemichannels represent a powerful medium for intercellular communication between astrocytes and neurons, their function in physiological conditions remains largely unexplored. Our results show that in the olfactory bulb, connexin 43 hemichannel function is promoted by neuronal activity and, in turn, modulates neuronal network slow oscillations. This novel mechanism of neuroglial interaction could influence olfactory information processing by directly impacting the output of the olfactory bulb.

Integrating temperature with odor processing in the olfactory bulb.

Temperature perception has long been classified as a somesthetic function solely. However, in recent years several studies brought evidence that temperature perception also takes place in the olfactory system of rodents. Temperature has been described as an effective stimulus for sensory neurons of the Grueneberg ganglion located at the entrance of the nose. Here, we investigate whether a neuronal trace of temperature stimulation can be observed in the glomeruli and mitral cells of the olfactory bulb, using calcium imaging and fast line-scanning microscopy. We show in the Xenopus tadpole system that the γ-glomerulus, which receives input from olfactory neurons, is highly sensitive to temperature drops at the olfactory epithelium. We observed that thermo-induced activity in the γ-glomerulus is conveyed to the mitral cells innervating this specific neuropil. Surprisingly, a substantial number of thermosensitive mitral cells were also chemosensitive. Moreover, we report another unique feature of the γ-glomerulus: it receives ipsilateral and contralateral afferents. The latter fibers pass through the contralateral bulb, cross the anterior commissure, and then run to the ipsilateral olfactory bulb, where they target the γ-glomerulus. Temperature drops at the contralateral olfactory epithelium also induced responses in the γ-glomerulus and in mitral cells. Temperature thus appears to be a relevant physiological input to the Xenopus olfactory system. Each olfactory bulb integrates and codes temperature signals originating from receptor neurons of the ipsilateral and contralateral nasal cavities. Finally, temperature and chemical information is processed in shared cellular networks.

Parallel processing of afferent olfactory sensory information.

KEY POINTS: The functional synaptic connectivity between olfactory receptor neurons and principal cells within the olfactory bulb is not well understood. One view suggests that mitral cells, the primary output neuron of the olfactory bulb, are solely activated by feedforward excitation. Using focal, single glomerular stimulation, we demonstrate that mitral cells receive direct, monosynaptic input from olfactory receptor neurons. Compared to external tufted cells, mitral cells have a prolonged afferent-evoked EPSC, which serves to amplify the synaptic input. The properties of presynaptic glutamate release from olfactory receptor neurons are similar between mitral and external tufted cells. Our data suggest that afferent input enters the olfactory bulb in a parallel fashion. ABSTRACT: Primary olfactory receptor neurons terminate in anatomically and functionally discrete cortical modules known as olfactory bulb glomeruli. The synaptic connectivity and postsynaptic responses of mitral and external tufted cells within the glomerulus may involve both direct and indirect components. For example, it has been suggested that sensory input to mitral cells is indirect through feedforward excitation from external tufted cells. We also observed feedforward excitation of mitral cells with weak stimulation of the olfactory nerve layer; however, focal stimulation of an axon bundle entering an individual glomerulus revealed that mitral cells receive monosynaptic afferent inputs. Although external tufted cells had a 4.1-fold larger peak EPSC amplitude, integration of the evoked currents showed that the synaptic charge was 5-fold larger in mitral cells, reflecting the prolonged response in mitral cells. Presynaptic afferents onto mitral and external tufted cells had similar quantal amplitude and release probability, suggesting that the larger peak EPSC in external tufted cells was the result of more synaptic contacts. The results of the present study indicate that the monosynaptic afferent input to mitral cells depends on the strength of odorant stimulation. The enhanced spiking that we observed in response to brief afferent input provides a mechanism for amplifying sensory information and contrasts with the transient response in external tufted cells. These parallel input paths may have discrete functions in processing olfactory sensory input.

A computational framework for temporal sharpening of stimulus input in the olfactory system.

The olfactory bulb glomerulus is a dense amalgamation of many unique and interconnected cell types. The mechanisms by which these neurons transform incoming information from the sensory periphery have been extensively studied but often with conflicting findings. A recent study by Carey et al. (J Neurophysiol 113: 3 112-3129, 2015) details the computational framework for parallel modes of temporal refinement of stimulus input to the olfactory system mediated by local neurons within individual glomeruli.

Presynaptic T-type Ca2+ channels modulate dendrodendritic mitral-mitral and mitral-periglomerular connections in mouse olfactory bulb.

Mitral cells express low-voltage activated Cav3.3 channels on their distal apical tuft dendrites (McKay et al., 2006; Johnston and Delaney, 2010). They also discharge Na(+)-dependent dendritic action potentials and release glutamate from these dendrites. Around resting membrane potentials, between -65 and -50 mV, Cav3.x channels are a primary determinant of cytoplasmic [Ca(2+)]. In this study using C57 mice, we present evidence that subthreshold Cav3.x-mediated Ca(2+) influx modulates action potential evoked transmitter release and directly drives asynchronous release from distal tuft dendrites. Presynaptic hyperpolarization and selective block of Cav3.x channels with Z941 (Tringham et al., 2012) reduce mitral-to-mitral EPSP amplitude, increase the coefficient of variation of EPSPs, and increase paired-pulse ratios, consistent with a reduced probability of transmitter release. Both hyperpolarization and Cav3.x channel blockade reduce steady-state cytoplasmic [Ca(2+)] in the tuft dendrite without reducing action potential evoked Ca(2+) influx, suggesting that background [Ca(2+)] modulates evoked release. We demonstrate that Cav3.x-mediated Ca(2+) influx from even one mitral cell at membrane potentials between -65 and -50 mV is sufficient to produce feedback inhibition from periglomerular neurons. Deinactivation of Cav3.x channels by hyperpolarization increases T-type Ca(2+) influx upon repolarization and increases feedback inhibition to produce subthreshold modulation of the mitral-periglomerular reciprocal circuit.

Notch1 activity in the olfactory bulb is odour-dependent and contributes to olfactory behaviour.

Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.

Regenerative capacity of bulbar projection neurons during development: a quantitative neuronal analysis with functional correlation.

Critical periods and degrees of regeneration in injured olfactory bulbar projection neurons (mitral cells) were examined in adult rats whose lateral olfactory tracts (LOTs) were transected at different postnatal (P) days. After the LOTs were transected at P7, P10, and P14, a retrograde fluorescent tracer, Fluoro-Gold (FG), was injected into the posterior olfactory cortex (the olfactory tubercle and the piriform cortex), a target brain region of mitral cells, 5 weeks after the transection. FG (+) mitral cells were observed in P7 LOT-transected bulbs and some of P10 LOT-transected bulbs but not in P14 LOT-transected bulbs. Neuron numbers of regenerated FG (+) mitral cells in P2 LOT-transected adult rats decreased to approximately 70% of the normal values (actually counted number: 804±46; stereologically estimated number: 49 700±4300), and 100% of these rats were demonstrated to exhibit olfactory discriminative ability in our previous study. Meanwhile, the numbers in P7 LOT-transected adult rats further decreased to approximately 40% of the normal values, and 78% of these rats showed olfactory discriminative ability. We conclude that the critical periods of spontaneous regeneration of the LOT are between P0 and P10 and that the proportions of regenerated mitral cells decreased as rats became older.

Sniff rhythm-paced fast and slow gamma-oscillations in the olfactory bulb: relation to tufted and mitral cells and behavioral states.

Odor signals are conveyed from the olfactory bulb (OB) to the olfactory cortex by two types of projection neurons, tufted cells and mitral cells, which differ in signal timing and firing frequency in response to odor inhalation. Whereas tufted cells respond with early-onset high-frequency burst discharges starting at the middle of the inhalation phase of sniff, mitral cells show odor responses with later-onset lower-frequency burst discharges. Since odor inhalation induces prominent gamma-oscillations of local field potentials (LFPs) in the OB during the transition period from inhalation to exhalation that accompany synchronized spike discharges of tufted cells and mitral cells, we addressed the question of whether the odor-induced gamma-oscillations encompass two distinct gamma-oscillatory sources, tufted cell and mitral cell subsystems, by simultaneously recording the sniff rhythms and LFPs in the OB of freely behaving rats. We observed that individual sniffs induced nested gamma-oscillations with two distinct parts during the inhalation-exhalation transition period: early-onset fast gamma-oscillations followed by later-onset slow gamma-oscillations. These results suggest that tufted cells carry odor signals with early-onset fast gamma-synchronization at the early phase of sniff, whereas mitral cells send them with later-onset slow gamma-synchronization. We also observed that each sniff typically induced both fast and slow gamma-oscillations during awake, whereas respiration during slow-wave sleep and rapid-eye-movement sleep failed to induce these oscillations. These results suggest that behavioral states regulate the generation of sniff rhythm-paced fast and slow gamma-oscillations in the OB.

Microglia Are Dispensable for Developmental Dendrite Pruning of Mitral Cells in Mice.

During early development, neurons in the brain often form excess synaptic connections. Later, they strengthen some connections while eliminating others to build functional neuronal circuits. In the olfactory bulb, a mitral cell initially extends multiple dendrites to multiple glomeruli but eventually forms a single primary dendrite through the activity-dependent dendrite pruning process. Recent studies have reported that microglia facilitate synapse pruning during the circuit remodeling in some systems. It has remained unclear whether microglia are involved in the activity-dependent dendrite pruning in the developing brains. Here, we examined whether microglia are required for the developmental dendrite pruning of mitral cells in mice. To deplete microglia in the fetal brain, we treated mice with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622, from pregnancy. Microglia were reduced by >90% in mice treated with PLX5622. However, dendrite pruning of mitral cells was not significantly affected. Moreover, we found no significant differences in the number, density, and size of excitatory synapses formed in mitral cell dendrites. We also found no evidence for the role of microglia in the activity-dependent dendrite remodeling of layer 4 (L4) neurons in the barrel cortex. In contrast, the density of excitatory synapses (dendritic spines) in granule cells in the olfactory bulb was significantly increased in mice treated with PLX5622 at postnatal day (P) 6, suggesting a role for the regulation of dendritic spines. Our results indicate that microglia do not play a critical role in activity-dependent dendrite pruning at the neurite level during early postnatal development in mice.

Dense and Sparse Labelling of Mitral Cells by Oral and Intraperitoneal Routes of Tamoxifen Administration.

Cell type-specific labelling and manipulation using Cre-driver lines have become integral to analyses of neuronal circuits in the brain. To study how mitral cells of the olfactory bulb process olfactory information and how they contribute to behavior, an inducible Cre-driver line, Lbhd2-CreERT2, can be used. In this chapter, we describe two methods for administering tamoxifen. The first method achieves a dense recombination pattern using tamoxifen-containing food, while the second method involving an intraperitoneal injection is suited for sparse labelling.