Optogenetic activation of dorsal raphe serotonin neurons induces brain-wide activation.

Serotonin is a neuromodulator that affects multiple behavioral and cognitive functions. Nonetheless, how serotonin causes such a variety of effects via brain-wide projections and various receptors remains unclear. Here we measured brain-wide responses to optogenetic stimulation of serotonin neurons in the dorsal raphe nucleus (DRN) of the male mouse brain using functional MRI with an 11.7 T scanner and a cryoprobe. Transient activation of DRN serotonin neurons caused brain-wide activation, including the medial prefrontal cortex, the striatum, and the ventral tegmental area. The same stimulation under anesthesia with isoflurane decreased brain-wide activation, including the hippocampal complex. These brain-wide response patterns can be explained by DRN serotonergic projection topography and serotonin receptor expression profiles, with enhanced weights on 5-HT1 receptors. Together, these results provide insight into the DR serotonergic system, which is consistent with recent discoveries of its functions in adaptive behaviors.

Quantum optical tomography based on time-resolved and mode-selective single-photon detection by femtosecond up-conversion.

We developed an optical time-of-flight measurement system using a time-resolved and mode-selective up-conversion single-photon detector for acquiring tomographic images of a mouse brain. The probe and pump pulses were spectrally carved from a 100-femtosecond mode-locked fiber laser at 1556 nm using 4f systems, so that their center wavelengths were situated at either side of the phase matching band separated by 30 nm. We demonstrated a sensitivity of 111 dB which is comparable to that of shot-noise-limited optical coherence tomography and an axial resolution of 57 µm (a refractive index of 1.37) with 380 femtosecond probe and pump pulses whose average powers were 1.5 mW and 30 µW, respectively. The proposed technique will open a new way of non-contact and non-invasive three-dimensional structural imaging of biological specimens with ultraweak optical irradiation.

Slow-rising and fast-falling dopaminergic dynamics jointly adjust negative prediction error in the ventral striatum.

The greater the reward expectations are, the more different the brain's physiological response will be. Although it is well-documented that better-than-expected outcomes are encoded quantitatively via midbrain dopaminergic (DA) activity, it has been less addressed experimentally whether worse-than-expected outcomes are expressed quantitatively as well. We show that larger reward expectations upon unexpected reward omissions are associated with the preceding slower rise and following larger decrease (DA dip) in the DA concentration at the ventral striatum of mice. We set up a lever press task on a fixed ratio (FR) schedule requiring five lever presses as an effort for a food reward (FR5). The mice occasionally checked the food magazine without a reward before completing the task. The percentage of this premature magazine entry (PME) increased as the number of lever presses approached five, showing rising expectations with increasing proximity to task completion, and hence greater reward expectations. Fibre photometry of extracellular DA dynamics in the ventral striatum using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m ) revealed that the slow increase and fast decrease in DA levels around PMEs were correlated with the PME percentage, demonstrating a monotonic relationship between the DA dip amplitude and degree of expectations. Computational modelling of the lever press task implementing temporal difference errors and state transitions replicated the observed correlation between the PME frequency and DA dip amplitude in the FR5 task. Taken together, these findings indicate that the DA dip amplitude represents the degree of reward expectations monotonically, which may guide behavioural adjustment.

Pupil Dynamics-derived Sleep Stage Classification of a Head-fixed Mouse Using a Recurrent Neural Network.

The standard method for sleep state classification is thresholding the amplitudes of electroencephalography (EEG) and electromyography (EMG) data, followed by manual correction by an expert. Although popular, this method has some shortcomings: (1) the time-consuming manual correction by human experts is sometimes a bottleneck hindering sleep studies, (2) EEG electrodes on the skull interfere with wide-field imaging of the cortical activity of a head-fixed mouse under a microscope, (3) invasive surgery to fix the electrodes on the thin mouse skull risks brain tissue injury, and (4) metal electrodes for EEG and EMG recording are difficult to apply to some experimental apparatus such as that for functional magnetic resonance imaging. To overcome these shortcomings, we propose a pupil dynamics-based vigilance state classification method for a head-fixed mouse using a long short-term memory (LSTM) model, a variant of a recurrent neural network, for multi-class labeling of NREM, REM, and WAKE states. For supervisory hypnography, EEG and EMG recording were performed on head-fixed mice. This setup was combined with left eye pupillometry using a USB camera and a markerless tracking toolbox, DeepLabCut. Our open-source LSTM model with feature inputs of pupil diameter, pupil location, pupil velocity, and eyelid opening for 10 s at a 10 Hz sampling rate achieved vigilance state estimation with a higher classification performance (macro F1 score, 0.77; accuracy, 86%) than a feed-forward neural network. Findings from a diverse range of pupillary dynamics implied possible subdivision of the vigilance states defined by EEG and EMG. Pupil dynamics-based hypnography can expand the scope of alternatives for sleep stage scoring of head-fixed mice.

A reinforcement learning model with choice traces for a progressive ratio schedule.

The progressive ratio (PR) lever-press task serves as a benchmark for assessing goal-oriented motivation. However, a well-recognized limitation of the PR task is that only a single data point, known as the breakpoint, is obtained from an entire session as a barometer of motivation. Because the breakpoint is defined as the final ratio of responses achieved in a PR session, variations in choice behavior during the PR task cannot be captured. We addressed this limitation by constructing four reinforcement learning models: a simple Q-learning model, an asymmetric model with two learning rates, a perseverance model with choice traces, and a perseverance model without learning. These models incorporated three behavioral choices: reinforced and non-reinforced lever presses and void magazine nosepokes, because we noticed that male mice performed frequent magazine nosepokes during PR tasks. The best model was the perseverance model, which predicted a gradual reduction in amplitudes of reward prediction errors (RPEs) upon void magazine nosepokes. We confirmed the prediction experimentally with fiber photometry of extracellular dopamine (DA) dynamics in the ventral striatum of male mice using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m). We verified application of the model by acute intraperitoneal injection of low-dose methamphetamine (METH) before a PR task, which increased the frequency of magazine nosepokes during the PR session without changing the breakpoint. The perseverance model captured behavioral modulation as a result of increased initial action values, which are customarily set to zero and disregarded in reinforcement learning analysis. Our findings suggest that the perseverance model reveals the effects of psychoactive drugs on choice behaviors during PR tasks.

Oscillatory population-level activity of dorsal raphe serotonergic neurons is inscribed in sleep structure.

Dorsal raphe (DR) 5-HT neurons regulate sleep-wake transitions. Previous studies demonstrated that single-unit activity of DR 5-HT neurons is high during wakefulness, decreases during non-rapid eye movement (NREM) sleep, and ceases during rapid eye movement (REM) sleep. However, characteristics of the population-level activity of DR 5-HT neurons, which influence the entire brain, are largely unknown. Here, we measured population activities of 5-HT neurons in the male and female mouse DR across the sleep-wake cycle by ratiometric fiber photometry. We found a slow oscillatory activity of compound intracellular Ca2+ signals during NREM sleep. The trough of the concave 5-HT activity increased across sleep progression, but 5-HT activity always returned to that seen during the wake period. When the trough reached a minimum and remained there, REM sleep was initiated. We also found a unique coupling of the oscillatory 5-HT activity and wide-band EEG power fluctuation. Furthermore, optogenetic activation of 5-HT neurons during NREM sleep triggered a high EMG power and induced wakefulness, demonstrating a causal role of 5-HT neuron activation. Optogenetic inhibition induced REM sleep or sustained NREM, with an EEG power increase and EEG fluctuation, and pharmacological silencing of 5-HT activity using a selective serotonin reuptake inhibitor led to sustained NREM, with an EEG power decrease and EEG fluctuation. These inhibitory manipulations supported the association between oscillatory 5-HT activity and EEG fluctuation. We propose that NREM sleep is not a monotonous state, but rather it contains dynamic changes that coincide with the oscillatory population-level activity of DR 5-HT neurons.SIGNIFICANT STATEMENTPrevious studies have demonstrated single-cell 5-HT neuronal activity across sleep-wake conditions. However, population-level activities of these neurons are not well understood. We monitored dorsal raphe (DR) 5-HT population activity using a fiber photometry system in mice and found that activity was highest during wakefulness and lowest during rapid eye movement (REM) sleep. Surprisingly, during non-REM sleep, the 5-HT population activity decreased with an oscillatory pattern, coinciding with EEG fluctuations. EEG fluctuations persisted when DR 5-HT neuron activity was silenced by either optogenetic or pharmacological interventions during non-REM sleep, suggesting an association between the two. Although oscillatory DR 5-HT neuron activity did not generate EEG fluctuations, it provides evidence that non-REM sleep exhibits at least binary states.

[The Logic of Scientific Reasoning in Peer Review Process].

We outlined the logical argumentation of scientific reasoning, focusing on peer-reviewed rebuttal letters. Initially, we compared the argumentation in the peer review process with that in writing papers, and distinguished between commonly used argumentation and those that are characteristic of rebuttal letters. First, commonly used forms of logical argumentation, such as deductive and inductive argumentation, are introduced, followed by examples of typical errors in reasoning. Subsequently, we explain the forms of logical argumentation characteristic of the peer review process, including the issue of incommensurability and the error of double standards. This article aims to improve our skills in responding to peer-review comments by being more aware of the logical argumentation used in peer review.

Dysfunction of parvalbumin-expressing cells in the thalamic reticular nucleus induces cortical spike-and-wave discharges and an unconscious state.

Spike-and-wave discharges and an accompanying loss of consciousness are hallmarks of absence seizure, which is a childhood generalized epilepsy disorder. In absence seizure, dysfunction of the cortico-thalamo-cortico circuitry is thought to engage in abnormal cortical rhythms. Previous studies demonstrated that the thalamic reticular nucleus has a critical role in the formation of normal cortical rhythms; however, whether thalamic reticular nucleus dysfunction leads directly to abnormal rhythms, such as epilepsy, is largely unknown. We found that expressing the inhibitory opsin, archaerhodopsin, including in the thalamic reticular nucleus, caused abnormal cortical rhythms in Pvalb-tetracycline transactivator::tetO-ArchT (PV-ArchT) double transgenic mice. We validated the PV-ArchT line as a new mouse model of absence seizure through physiological and pharmacological analyses, as well as through examining their behavioural features. We then discovered that archaerhodopsin expression exclusively in thalamic reticular nucleus parvalbumin-positive neurons was sufficient to induce cortical spike-and-wave discharges using adeno-associated virus-mediated thalamic reticular nucleus targeting. Furthermore, we found that archaerhodopsin expression impaired rebound burst firing and T-current in thalamic reticular nucleus parvalbumin-positive cells by slice physiology. Although T-current in the thalamic reticular nucleus was impaired, the T-current blocker ethosuximide still had a therapeutic effect in PV-ArchT mice, suggesting a gain of function of T-type calcium channels in this absence seizure model. However, we did not find any over- or misexpression of T-type calcium channel genes in the thalamus or the cortex. Thus, we demonstrated that thalamic reticular nucleus dysfunction led to an absence seizure-like phenotype in mice. In a final set of experiments, we showed that the archaerhodopsin-mediated absence seizure-like phenotype disappeared after the removal of archaerhodopsin by using a time-controllable transgenic system. These data may provide a hint as to why many absence seizures naturally regress.

Rhythmic activation of excitatory neurons in the mouse frontal cortex improves the prefrontal cortex-mediated cognitive function.

The prefrontal cortex (PFC) plays essential roles in cognitive processes. Previous studies have suggested the layer and the cell type-specific activation for cognitive enhancement. However, the mechanism by which a temporal pattern of activation affects cognitive function remains to be elucidated. Here, we investigated whether the specific activation of excitatory neurons in the superficial layers mainly in the PFC according to a rhythmic or nonrhythmic pattern could modulate the cognitive functions of normal mice. We used a C128S mutant of channelrhodopsin 2, a step function opsin, and administered two light illumination patterns: (i) alternating pulses of blue and yellow light for rhythmic activation or (ii) pulsed blue light only for nonrhythmic activation. Behavioral analyses were performed to compare the behavioral consequences of these two neural activation patterns. The alternating blue and yellow light pulses, but not the pulsed blue light only, significantly improved spatial working memory and social recognition without affecting motor activity or the anxiety level. These results suggest that the rhythmic, but not the nonrhythmic, activation could enhance cognitive functions. This study indicates that not only the population of neurons that are activated but also the pattern of activation plays a crucial role in the cognitive enhancement.

Optical manipulation of local cerebral blood flow in the deep brain of freely moving mice.

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.

Flexible annotation atlas of the mouse brain: combining and dividing brain structures of the Allen Brain Atlas while maintaining anatomical hierarchy.

A brain atlas is necessary for analyzing structure and function in neuroimaging research. Although various annotation volumes (AVs) for the mouse brain have been proposed, it is common in magnetic resonance imaging (MRI) of the mouse brain that regions-of-interest (ROIs) for brain structures (nodes) are created arbitrarily according to each researcher's necessity, leading to inconsistent ROIs among studies. One reason for such a situation is the fact that earlier AVs were fixed, i.e. combination and division of nodes were not implemented. This report presents a pipeline for constructing a flexible annotation atlas (FAA) of the mouse brain by leveraging public resources of the Allen Institute for Brain Science on brain structure, gene expression, and axonal projection. A mere two-step procedure with user-specified, text-based information and Python codes constructs FAA with nodes which can be combined or divided objectively while maintaining anatomical hierarchy of brain structures. Four FAAs with total node count of 4, 101, 866, and 1381 were demonstrated. Unique characteristics of FAA realized analysis of resting-state functional connectivity (FC) across the anatomical hierarchy and among cortical layers, which were thin but large brain structures. FAA can improve the consistency of whole brain ROI definition among laboratories by fulfilling various requests from researchers with its flexibility and reproducibility.

Chronic social defeat stress impairs goal-directed behavior through dysregulation of ventral hippocampal activity in male mice.

Chronic stress is a risk factor for a variety of psychiatric disorders, including depression. Although impairments to motivated behavior are a major symptom of clinical depression, little is known about the circuit mechanisms through which stress impairs motivation. Furthermore, research in animal models for depression has focused on impairments to hedonic aspects of motivation, whereas patient studies suggest that impairments to appetitive, goal-directed motivation contribute significantly to motivational impairments in depression. Here, we characterized goal-directed motivation in repeated social defeat stress (R-SDS), a well-established mouse model for depression in male mice. R-SDS impaired the ability to sustain and complete goal-directed behavior in a food-seeking operant lever-press task. Furthermore, stress-exposed mice segregated into susceptible and resilient subpopulations. Interestingly, susceptibility to stress-induced motivational impairments was unrelated to stress-induced social withdrawal, another prominent effect of R-SDS in mouse models. Based on evidence that ventral hippocampus (vHP) modulates sustainment of goal-directed behavior, we monitored vHP activity during the task using fiber photometry. Successful task completion was associated with suppression of ventral hippocampal neural activity. This suppression was diminished after R-SDS in stress-susceptible but not stress-resilient mice. The serotonin selective reuptake inhibitor (SSRI) escitalopram and ketamine both normalized vHP activity during the task and restored motivated behavior. Furthermore, optogenetic vHP inhibition was sufficient to restore motivated behavior after stress. These results identify vHP hyperactivity as a circuit mechanism of stress-induced impairments to goal-directed behavior and a putative biomarker that is sensitive to antidepressant treatments and that differentiates susceptible and resilient individuals.

[Global Circuit of the Brain through the Thalamus].

What is the physiological mechanism of the harmonic dynamics of whole-brain activity? As a clue to answering this question, I have summarized the anatomical findings of global brain circuits, including the basal ganglia and cerebellum. The thalamus always appeared in these circuits as a collection point for all information. The "parallel circuit" seemed to be a basic structure of the global brain circuits. The interaction between parallel circuits was suggested to be achieved by convergence and divergence of information through the parallel circuits, and by switching between them.

Chd8 mutation in oligodendrocytes alters microstructure and functional connectivity in the mouse brain.

CHD8 encodes a chromatin-remodeling factor and is one of the most recurrently mutated genes in individuals with autism spectrum disorder (ASD). Although we have recently shown that mice heterozygous for Chd8 mutation manifest myelination defects and ASD-like behaviors, the detailed mechanisms underlying ASD pathogenesis have remained unclear. Here we performed diffusion tensor imaging (DTI) and resting-state functional magnetic resonance imaging (rsfMRI) in oligodendrocyte lineage-specific Chd8 heterozygous mutant mice. DTI revealed that ablation of Chd8 specifically in oligodendrocytes of mice was associated with microstructural changes of specific brain regions including the cortex and striatum. The extent of these changes in white matter including the corpus callosum and fornix was correlated with total contact time in the reciprocal social interaction test. Analysis with rsfMRI revealed changes in functional brain connectivity in the mutant mice, and the extent of such changes in the cortex, hippocampus, and amygdala was also correlated with the change in social interaction. Our results thus suggest that changes in brain microstructure and functional connectivity induced by oligodendrocyte dysfunction might underlie altered social interaction in mice with oligodendrocyte-specific CHD8 haploinsufficiency.

Diffusion functional MRI reveals global brain network functional abnormalities driven by targeted local activity in a neuropsychiatric disease mouse model.

Diffusion functional magnetic resonance imaging (DfMRI) has been proposed as an alternative functional imaging method to detect brain activity without confounding hemodynamic effects. Here, taking advantage of this DfMRI feature, we investigated abnormalities of dynamic brain function in a neuropsychiatric disease mouse model (glial glutamate transporter-knockdown mice with obsessive-compulsive disorder [OCD]-related behavior). Our DfMRI approaches consisted of three analyses: resting state brain activity, functional connectivity, and propagation of neural information. We detected hyperactivation and biased connectivity across the cortico-striatal-thalamic circuitry, which is consistent with known blood oxygen-level dependent (BOLD)-fMRI patterns in OCD patients. In addition, we performed ignition-driven mean integration (IDMI) analysis, which combined activity and connectivity analyses, to evaluate neural propagation initiated from brain activation. This analysis revealed an unbalanced distribution of neural propagation initiated from intrinsic local activation to the global network, while these were not detected by the conventional method with BOLD-fMRI. This abnormal function detected by DfMRI was associated with OCD-related behavior. Together, our comprehensive DfMRI approaches can successfully provide information on dynamic brain function in normal and diseased brains.

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep-wake states.

Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5'-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

Thalamic reticular nucleus in the thalamocortical loop.

Dynamic binding of different brain areas is critical for various cognitive functions. The thalamic reticular nucleus (TRN) is a GABAergic nucleus that constrains information flow through thalamocortical loop by providing inhibitory innervation to the thalamus. In this review, I summarize anatomical and single-cell-level physiological studies of the rodent TRN. Diversity and heterogeneity of TRN neurons in terms of axonal innervation, molecular expression, and physiological characteristics are described. I also outline thalamocortical and cortico-cortical connections with emphasis on interaction with the TRN. In summary, it is proposed that functional connectivity among brain regions are modulated with gating of transthalamic information flow by the TRN.

Common functional networks in the mouse brain revealed by multi-centre resting-state fMRI analysis.

Preclinical applications of resting-state functional magnetic resonance imaging (rsfMRI) offer the possibility to non-invasively probe whole-brain network dynamics and to investigate the determinants of altered network signatures observed in human studies. Mouse rsfMRI has been increasingly adopted by numerous laboratories worldwide. Here we describe a multi-centre comparison of 17 mouse rsfMRI datasets via a common image processing and analysis pipeline. Despite prominent cross-laboratory differences in equipment and imaging procedures, we report the reproducible identification of several large-scale resting-state networks (RSN), including a mouse default-mode network, in the majority of datasets. A combination of factors was associated with enhanced reproducibility in functional connectivity parameter estimation, including animal handling procedures and equipment performance. RSN spatial specificity was enhanced in datasets acquired at higher field strength, with cryoprobes, in ventilated animals, and under medetomidine-isoflurane combination sedation. Our work describes a set of representative RSNs in the mouse brain and highlights key experimental parameters that can critically guide the design and analysis of future rodent rsfMRI investigations.

Visualization of myelinated fiber bundles orientation during brain slice preparation by reflection polarized light microscopy.

Polarized light imaging (PLI) enables detecting the orientation of myelinated axon bundles in brain slices at microscopic resolution without histological staining. However, standard PLI requires labor-intensive procedures such as mounting brain cryosections on slide glasses. We developed an optical system that does not require a mounting procedure for PLI. Specifically, we developed an optical system to perform PLI in reflection mode (rPLI) instead of employing transmitted light as in standard PLI. We integrated this rPLI system with a conventional vibratome slicer whose cutting blade surface is a mirror. This combination allowed PLI measurements directly during the slicing procedure at room temperature. Thus, mounting procedure for PLI is not necessary. As a proof-of-concept experiment, a perfusion-fixed brain of a mouse was embedded in gelatin-containing agar and cut serially at 40~200 µm intervals. The slicing procedure was temporarily halted after each cut to capture the PLI images of the slice on the reflecting blade surface while the slice was still held up by the agar block. The orientation of the fiber bundle estimated with this method agreed with the results obtained from previous reports. Combination of a popular vibratome slicer and our rPLI system that uses versatile and inexpensive optical components would increase popularity of PLI and facilitates connectome studies at microscopic resolution. RESEARCH HIGHLIGHTS: Polarized light imaging (PLI) of brain slices was realized by using reflected light (rPLI) instead of transmitted light. The rPLI method allows detecting the myelinated fiber bundle orientation during slice preparation.

Assessing cortical plasticity after spinal cord injury by using resting-state functional magnetic resonance imaging in awake adult mice.

Neural connectivity has recently been shown to be altered after spinal cord injury (SCI) not only in the spinal cord but also in the brain. However, to date, no studies have analyzed the functional alterations after SCI in various areas of the cerebral cortex over time. To examine the plasticity of the neural connectivity in the brain after SCI, we performed resting-state functional magnetic resonance imaging (rs-fMRI) in awake adult mice pre- and post-SCI. After a complete thoracic SCI, the functional connectivity between the primary motor (MOp) and primary sensory (SSp) areas was significantly decreased during the chronic phase. In contrast, the connectivity between the MOp and motivation area was increased. Thus, impairments in sensory and motor connections after SCI led to a time-dependent compensatory upregulation of "motor functional motivation". Moreover, the functional connectivity between the SSp and pain-related areas, such as the caudoputamen (CP) and the anterior cingulate area (ACA), was strengthened during the chronic phase, thus suggesting that rs-fMRI can indicate the presence of neuropathic pain after SCI. Therefore, rs-fMRI is a useful tool for revealing the pathological changes that occur in the brain after SCI.