Biology subjects

Fukunaga, I.

Publications and source records attributed to Fukunaga, I..

13 recordsLinked to original sources

bioRxiv2026

Molecularly defined olfactory bulb output neurons exhibit selective yet overlapping projections

Early sensory processing decomposes rich signals into elemental features that can be conveyed through parallel neural pathways defined by cell types, with unique local circuit organization and long-range projection targets. Outputs from the olfactory bulb are typically categorized into two broad pathways corresponding to mitral and tufted cells. Previous high-throughput molecular profiling identified finer classes, but their morphology, projection patterns, and downstream convergence remain unclear. Here, we use an intersectional genetic targeting method with two Cre driver lines, retrograde projection mapping from six cortical targets, and RiboTag-based sequencing to study the subtypes of olfactory bulb output neurons. We found that retrograde labeling from the same cortical area recruited morphologically different OB neurons depending on the Cre driver used, suggesting a cortical area can be targeted by several subtypes. Comparing the marker expression, morphology, and downstream targets, we found that M1 neurons are densely labelled from the cortical amygdala, whereas M2 neurons project widely throughout the olfactory cortex but can be selectively recruited from the posterior piriform cortex. T1 cells differ from external tufted subtypes in their projections to the cap of the olfactory tubercle, while ET2 cells preferentially target the anterior olfactory nucleus pars externa. Electrophysiology of labelled neurons with different morphology and projection patterns differ in their intrinsic and functional properties. Further, our data suggest that extracellular recording methods may underrepresent mitral cells. Together, these findings reveal that molecularly defined olfactory bulb output neuron subtypes form anatomically and physiologically distinct channels that route information to overlapping yet selective downstream cortical targets.

neuroscience↗

bioRxiv2026

Hierarchy in the mouse frontal cortex in mnemonic olfactory decision-making

The prefrontal cortex plays a critical role in integrating the memory of a recent experience to guide context-dependent decisions, yet how the finer, sub-steps of decision formation are physiologically implemented remains poorly understood. Using an olfactory delayed non-match-to-sample task with graded stimulus similarity in head-fixed mice, we examined how decisions emerge when a current sensory event and a memory of a recent event must be compared. With high-density extracellular recordings across the frontal cortex, we characterized odor-specific delay activity and decision-related signals. Interestingly, the secondary motor cortex showed minimal sensory coding, suggesting that areas engaged in mnemonic decision-making differ from those involved in simpler, stimulus-response decision-making in the rodent brain. Rather, there was a gradual emergence from sensory representations to choice-related activity, with intermediate regions showing choice modulations that retain stimulus sensitivity that arises with early timing, as well as balanced match vs. non-match selectivity. These results suggest that mnemonic decision-making is supported by a distributed frontal network in which sensory and choice signals are gradually integrated, rather than localized to a single comparator region.

neuroscience↗

bioRxiv2024

Decoding Olfactory Bulb Output: A Behavioral Assessment of Rate, Synchrony, and Respiratory Phase Coding

The olfactory system is a well-known model for studying the temporal encoding of sensory stimuli due to its rhythmic stimulus delivery through respiration. The sniff rhythm is considered critical to structuring the output of computation in the primary olfactory area, the olfactory bulb. Here, Neuropixels recording from awake, head-fixed mice confirmed that both the rate and sniff-locked spike timing are informative about odour identity. We tested the behavioural importance of these temporal features using simple closed-loop optogenetics embedded in custom behavioural paradigms. We found that mice perceive differences in evoked spike counts and discriminate between synchronous vs. asynchronous activations of the output neurons. Surprisingly, they failed to distinguish the timing of evoked activity relative to the sniff cycle. These results challenge the utility of internally generated rhythms as reference signals in the neural encoding of the environment.

neuroscience↗

bioRxiv2024

Body size interacts with the structure of the central nervous system: A multi-center in vivo neuroimaging study

Clinical research emphasizes the implementation of rigorous and reproducible study designs that rely on between-group matching or controlling for sources of biological variation such as subjects sex and age. However, corrections for body size (i.e. height and weight) are mostly lacking in clinical neuroimaging designs. This study investigates the importance of body size parameters in their relationship with spinal cord (SC) and brain magnetic resonance imaging (MRI) metrics. Data were derived from a cosmopolitan population of 267 healthy human adults (age 30.1{+/-}6.6 years old, 125 females). We show that body height correlated strongly or moderately with brain gray matter (GM) volume, cortical GM volume, total cerebellar volume, brainstem volume, and cross-sectional area (CSA) of cervical SC white matter (CSA-WM; 0.44[≤]r[≤]0.62). In comparison, age correlated weakly with cortical GM volume, precentral GM volume, and cortical thickness (-0.21[≥]r[≥]-0.27). Body weight correlated weakly with magnetization transfer ratio in the SC WM, dorsal columns, and lateral corticospinal tracts (-0.20[≥]r[≥]-0.23). Body weight further correlated weakly with the mean diffusivity derived from diffusion tensor imaging (DTI) in SC WM (r=-0.20) and dorsal columns (-0.21), but only in males. CSA-WM correlated strongly or moderately with brain volumes (0.39[≤]r[≤]0.64), and weakly with precentral gyrus thickness and DTI-based fractional anisotropy in SC dorsal columns and SC lateral corticospinal tracts (-0.22[≥]r[≥]-0.25). Linear mixture of sex and age explained 26{+/-}10% of data variance in brain volumetry and SC CSA. The amount of explained variance increased at 33{+/-}11% when body height was added into the mixture model. Age itself explained only 2{+/-}2% of such variance. In conclusion, body size is a significant biological variable. Along with sex and age, body size should therefore be included as a mandatory variable in the design of clinical neuroimaging studies examining SC and brain structure.

neuroscience↗

bioRxiv2023

Reward contingency modulates olfactory bulb output via pathway-dependent peri-somatic inhibition

Associating values to environmental cues is a critical aspect of learning from experiences, allowing animals to predict and maximise future rewards. Value-related signals in the brain were once considered a property of higher sensory regions, but its wide distribution across many brain regions is increasingly recognised. Here, we investigate how reward-related signals begin to be incorporated, mechanistically, at the earliest stage of olfactory processing, namely, in the olfactory bulb. In head-fixed mice performing Go/No-Go discrimination of closely related olfactory mixtures, rewarded odours evoke widespread inhibition in one class of output neurons, that is, in mitral cells but not tufted cells. The temporal characteristics of this reward-related inhibition suggest it is odour-driven, but it is also context-dependent since it is absent during pseudo-conditioning and pharmacological silencing of the piriform cortex. Further, the reward-related modulation is present in the somata but not in the apical dendritic tuft of mitral cells, suggesting an involvement of circuit component located deep in the olfactory bulb. Depth-resolved imaging from granule cell dendritic gemmules suggests that granule cells that target mitral cells receive a reward-related extrinsic drive. Our results support the notion that value-related modulation appears at the early stages of sensory processing and provide constraints on long-range and local circuit mechanisms.

neuroscience↗

bioRxiv2022

Effects of stimulus timing on the acquisition of an olfactory working memory task in head-fixed mice

Knowing what factors affect the acquisition of a behavioural task is central to understanding the mechanisms of learning and memory. It also has practical implications, as animal behavioural experiments used to probe cognitive functions often require long training durations. Delayed Match (or Non-Match)-to-Sample (DMS/DNMS) tasks are relatively complex tasks used to study working memory and sensory perception, but their use in the mouse remains hampered by the lengthy training involved. In this study, we assessed two aspects of stimulus timing on the acquisition of an olfactory DNMS task: how the sample-test odour delay durations and the reward timing affect the acquisition rate. We demonstrate that head-fixed mice learn to perform an olfactory DNMS task more quickly when the initial training uses a shorter sample-test odour delay without detectable loss of generalisability. Unexpectedly, we observed a slower task acquisition when the odour-reward interval was shorter. This effect was accompanied by a shortening of reaction times and more frequent sporadic licking. Analysis of this result using a drift-diffusion model indicated that a primary consequence of early reward delivery is a lower decision bound. Since an accurate performance with a lower decision bound requires greater discriminability in the sensory representations, this may underlie the slower learning rate with early reward arrival. Together, our results reflect the possible effects of stimulus timing on stimulus encoding and its consequence on the acquisition of a complex task.

neuroscience↗

bioRxiv2021

State-dependent linearisation of mixture representations by the olfactory bulb

Sensory systems are often tasked to analyse complex signals from the environment, separating relevant from irrelevant parts. This process of decomposing signals is challenging when a mixture of signals does not equal the sum of its parts, leading to an unpredictable corruption of signal patterns. In olfaction, nonlinear summation is prevalent at various stages of sensory processing. Here, we investigate how the olfactory system deals with binary mixtures of odours under different brain states, using two-photon imaging of olfactory bulb (OB) output neurons. Unlike previous studies using anaesthetised animals, we found that mixture summation is more linear in the early phase of evoked responses in awake, head-fixed mice performing an odour detection task, due to dampened responses. Despite this, and responses being more variable, decoding analyses indicated that the data from behaving mice was well discriminable. Curiously, the time course of decoding accuracy did not correlate strictly with the linearity of summation. Further, a comparison with naive mice indicated that learning to accurately perform the mixture detection task is not accompanied by more linear mixture summation. Finally, using a simulation, we demonstrate that, while saturating sublinearity tends to degrade the discriminability, the extent of the impairment may depend on other factors, including pattern decorrelation. Altogether, our results demonstrate that the mixture representation in the primary olfactory area is state-dependent, but the analytical perception may not strictly correlate with linearity in summation.

neuroscience↗

bioRxiv2020

Transcriptome analysis for the development of cell-type specific labeling to study olfactory circuits

In each sensory system of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioural repertoires. These often correspond to different cell types at some stage in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral and tufted cells (MCs and TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, little is known how these two types of projection neurons differ at the mRNA level. Here, we sought to identify genes that are differentially expressed between MCs and TCs, with an ultimate goal to generate a cell-type specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Despite many genes showing differential expressions, we identified only a few that were abundantly and consistently expressed only in MCs. After further validating these putative markers using in-situ hybridization, two genes, namely Pkib and Lbdh2, remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analysed the resulting recombination patterns. This analysis indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, as assessed by soma locations, projection patterns and sensory-evoked responses. Hence this line is a promising tool for future investigations of cell-type specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science.

neuroscience↗

bioRxiv2020

TGF-β inhibitor accelerates BMP4-induced cochlear gap junction formation during in vitro differentiation of embryonic stem cells

Mutations in the connexin 26 (CX26)/gap junction beta-2 (GJB2) gene are the most frequent cause of hereditary deafness worldwide. Using mouse induced pluripotent stem cells (iPSCs) and a BMP4 signal-based floating and adherent culture system, we recently produced in vitro responsible for GJB2-related deafness (CX26-gap junction plaque-forming cells, CX26GJCs). However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, presently uncharacterized factors may also induce CX26 gap junction (GJ) formation. A floating culture with embryonic stem cell (ESC) treatment and BMP4/TGF-{beta} inhibitor (SB431542:SB) has been shown to result in greater production of isolatable CX26-positive small vesicles (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified CX26GJCs. In the present study, we first demonstrated that SB accelerates BMP4 induced GJ formation during stem cell differentiation. By controlling the concentration and timing of SB supplementation with CX26+ vesicle purification, large-scale production of highly purified CX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

cell biology↗

bioRxiv2019

Mammalian olfaction is a high temporal bandwidth sense

Odours are transported in turbulent plumes resulting locally in highly fluctuating odour concentration (Celani et al., 2014; Murlis et al., 1992; Mylne and Mason, 1991; Shraiman and Siggia, 2000). Yet, whether mammals can make use of the ensuing temporal structure (Celani et al., 2014; Crimaldi and Koseff, 2001; Murlis et al., 1992; Mylne and Mason, 1991; Schmuker et al., 2016; Vickers, 2000) to extract information about the olfactory environment remains unknown. Here, we use dual-energy photoionisation recording with >300 Hz bandwidth to simultaneously determine odour concentrations of two odours in air. We show that temporal correlation of odour concentrations reliably predicts whether odorants emerge from the same or different sources in normal turbulent environments outside and in laboratory conditions. To replicate natural odour dynamics in a reproducible manner we developed a multichannel odour delivery device allowing presentation of several odours with 10 ms temporal resolution. Integrating this device in an automated operant conditioning system we demonstrate that mice can reliably discriminate the correlation structure of odours at frequencies of up to 40 Hz. Consistent with this finding, output neurons in the olfactory bulb show segregated responses depending on the correlation of odour stimuli with populations of 10s of neurons sufficient to reach behavioural performance. Our work thus demonstrates that mammals can perceive temporal structure in odour stimuli at surprisingly fast timescales. This in turn might be useful for key behavioural challenges (Jacobs, 2012) such as odour source separation (Hopfield, 1991), figure-ground separation (Rokni et al., 2014) or odour localisation (Vergassola et al., 2007; Vickers, 2000).

neuroscience↗

bioRxiv2018

Rapid task-dependent tuning of the mouse olfactory bulb

Adapting neural representation to rapidly changing behavioural demands is a key challenge for the nervous system. Here, we demonstrate that the output of the primary olfactory area, the mouse olfactory bulb, is already a target of dynamic and reproducible modulation. The modulation depends on the stimulus tuning of a given neuron, making olfactory responses more discriminable through selective amplification in a demand-specific way.

neuroscience↗

bioRxiv2017

Active sampling state dynamically enhances olfactory bulb odor representation

The olfactory bulb (OB) is the very first site of odor information processing, yet a wealth of contextual and learned information has been described in its activity. To investigate the mechanistic basis of contextual modulation, we use whole-cell recordings to measure odor responses across rapid (<30 min) learning episodes in identified mitral/tufted cells (MTCs). Across these learning episodes, we found that diverse response changes occur already during the first sniff cycle. Motivated mice develop active sniffing strategies across learning, and it is this change of active sampling state that dynamically modulates odor responses, resulting in enhanced discriminability and detectability of odor representation with learning. Evoking fast sniffing in different behavioral states demonstrates that response changes during active sampling exceed those predicted from purely feed-forward input. Finally, response changes are highly correlated in tufted cells, but not mitral cells, indicating cell-type specificity in the effect of active sampling, and resulting in increased odor detectability in the tufted and enhanced discriminability in the mitral cell population over the rapid learning episodes. Altogether, we show that active sampling state is a crucial component in modulating and enhancing olfactory bulb responsiveness on rapid timescales.

neuroscience↗

bioRxiv2017

Coherent olfactory bulb gamma oscillations arise from coupling independent columnar oscillators

Spike timing-based representations of sensory information depend on embedded dynamical frameworks within neuronal networks that establish the rules of local computation and interareal communication. Here, we investigated the dynamical properties of olfactory bulb circuitry in mice of both sexes using microelectrode array recordings from slice and in vivo preparations. Neurochemical activation or optogenetic stimulation of sensory afferents evoked persistent gamma oscillations in the local field potential. These oscillations arose from slower, GABA(A) receptor-independent intracolumnar oscillators coupled by GABA(A)-ergic synapses into a faster, broadly coherent network oscillation. Consistent with the theoretical properties of coupled-oscillator networks, the spatial extent of zero-phase coherence was bounded in slices by the reduced density of lateral interactions. The intact in vivo network, however, exhibited long-range lateral interactions that suffice in simulation to enable zero-phase gamma coherence across the olfactory bulb. The timing of action potentials in a subset of principal neurons was phase-constrained with respect to evoked gamma oscillations. Coupled-oscillator dynamics in olfactory bulb thereby enable a common clock, robust to biological heterogeneities, that is capable of supporting gamma-band spike synchronization and phase coding across the ensemble of activated principal neurons. New & NoteworthyOdor stimulation evokes rhythmic gamma oscillations in the field potential of the olfactory bulb, but the dynamical mechanisms governing these oscillations have remained unclear. Establishing these mechanisms is important, as they determine the biophysical capacities of the bulbar circuit to, for example, maintain zero-phase coherence across a spatially extended network, or coordinate the timing of action potentials in principal neurons. These properties in turn constrain and suggest hypotheses of sensory coding.

neuroscience↗