Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Odor-evoked layer-specific fMRI activities in the awake mouse olfactory bulb.

Awake rodent fMRI is increasingly common over the use of anesthesia since it permits behavioral paradigms and does not confound normal brain function or neurovascular coupling. It is well established that adequate acclimation to the loud fMRI environment and head fixation reduces stress in the rodents and allows for whole brain imaging with little contamination from motion. However, it is unknown whether high-resolution fMRI with increased susceptibility to motion and lower sensitivity can measure small, but spatially discrete, activations in awake mice. To examine this, we used contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI in the mouse olfactory bulb for its enhanced sensitivity and neural specificity. We determined that activation patterns in the glomerular layer to four different odors were spatially distinct and were consistent with previously established histological patterns. In addition, odor-evoked laminar activations were greatest in superficial layers that decreased with laminar depth, similar to previous observations. Interestingly, the fMRI response strengths in the granule cell layer were greater in awake mice than our previous anesthetized rat studies, suggesting that feedback neural activities were intact with wakefulness. We finally determined that fMRI signal changes to repeated odor exposure (i.e., olfactory adaptation) attenuated relatively more in the feedback granule cell layer compared to the input glomerular layer, which is consistent with prior observations. We, therefore, conclude that high-resolution CBVw fMRI can measure odor-specific activation patterns and distinguish changes in laminar activity of head and body restrained awake mice.

Heat Stroke Prevention in Hot Specific Occupational Environment Enhanced by Supervised Machine Learning with Personalized Vital Signs.

Recently, wet-bulb globe temperature (WBGT) has attracted a lot of attention as a useful index for measuring heat strokes even when core body temperature cannot be available for the prevention. However, because the WBGT is only valid in the vicinity of the WBGT meter, the actual ambient heat could be different even in the same room owing to ventilation, clothes, and body size, especially in hot specific occupational environments. To realize reliable heat stroke prevention in hot working places, we proposed a new personalized vital sign index, which is combined with several types of vital data, including the personalized heat strain temperature (pHST) index based on the temperature/humidity measurement to adjust the WBGT at the individual level. In this study, a wearable device was equipped with the proposed pHST meter, a heart rate monitor, and an accelerometer. Additionally, supervised machine learning based on the proposed personalized vital index was introduced to improve the prevention accuracy. Our developed system with the proposed vital sign index achieved a prevention accuracy of 85.2% in a hot occupational experiment in the summer season, where the true positive rate and true negative rate were 96.3% and 83.7%, respectively.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Chemical exchange sensitive MRI of glucose uptake using xylose as a contrast agent.

PURPOSE: Glucose and its analogs can be detected by CEST and chemical exchange spin-lock (CESL) MRI techniques, but sensitivity is still a bottleneck for human applications. Here, CESL and CEST sensitivity and the effect of injection on baseline physiology were evaluated for a glucose analog, xylose. METHODS: The CEST and CESL sensitivity were evaluated at 9.4 T in phantoms and by in vivo rat experiments with 0.5 and 1 g/kg xylose injections. Arterial blood glucose level was sampled before and after 1 g/kg xylose injection. The effect of injection on baseline neuronal activity was measured by electrophysiology data during injections of saline, xylose, and 2-deoxy-D-glucose. RESULTS: In phantoms, xylose shows similar chemical exchange sensitivity and pH-dependence with that of glucose. In rat experiments with a bolus injection, CESL shows higher sensitivity in the detection of xylose than CEST, and the sensitivity of xylose is much higher than glucose. Injection of xylose does not significantly affect blood glucose level and baseline neural activity for 1-g/kg and 0.6-g/kg doses, respectively. CONCLUSION: Due to its relatively high sensitivity and safety, xylose is a promising contrast agent for the study of glucose uptake.

α-Emitting cancer therapy using 211 At-AAMT targeting LAT1.

α-Methyl-l-tyrosine (AMT) has a high affinity for the cancer-specific l-type amino acid transporter 1 (LAT1). Therefore, we established an anti-cancer therapy, with 211 At-labeled α-methyl-l-tyrosine (211 At-AAMT) as a carrier of 211 At into tumors. 211 At-AAMT had high affinity for LAT1, inhibited tumor cell growth, and induced DNA double-stranded breaks in vitro. We evaluated the accumulation of 211 At-AAMT in vivo and the role of LAT1. Treatment with 0.4 MBq/mouse 211 At-AAMT inhibited tumor growth in the PANC-1 tumor model and 1 MBq/mouse 211 At-AAMT inhibited metastasis in the lung of the B16F10 metastasis model. Our results suggested that 211 At would be useful for anti-cancer therapy and that LAT1 is suitable as a target for radionuclide therapy.

Time-dependent spatial specificity of high-resolution fMRI: insights into mesoscopic neurovascular coupling.

High-resolution functional magnetic resonance imaging (fMRI) is becoming increasingly popular because of the growing availability of ultra-high magnetic fields which are capable of improving sensitivity and spatial resolution. However, it is debatable whether increased spatial resolutions for haemodynamic-based techniques, like fMRI, can accurately detect the true location of neuronal activity. We have addressed this issue in functional columns and layers of animals with haemoglobin-based optical imaging and different fMRI contrasts, such as blood oxygenation level-dependent, cerebral blood flow and cerebral blood volume fMRI. In this review, we describe empirical evidence primarily from our own studies on how well these fMRI signals are spatially specific to the neuronally active site and discuss insights into neurovascular coupling at the mesoscale. This article is part of the theme issue 'Key relationships between non-invasive functional neuroimaging and the underlying neuronal activity'.

Developments of real-time emittance monitors.

Under the upgrade program of an azimuthally varying field (AVF) cyclotron in progress at the Research Center for Nuclear Physics (RCNP), an emittance monitor is being developed to improve the beam injection efficiency from ion sources to the AVF cyclotron. In order to evaluate the quality of the beams extracted from ion sources quickly, we developed the Pepper-Pot type Emittance Monitor at the RCNP. After improving an analysis method for emittance estimation using LabVIEW, we achieved a measurement frequency of 4 Hz.

Development of an MeV proton generator using a 3He ion source at the Research Center for Nuclear Physics.

For simple applications, such as the calibration of a charged particle detector, a multi-MeV proton generator may be preferable to cyclotrons or electrostatic accelerators such as Van de Graaff generator. Thus, a proton generating system, consisting of an 18 GHz superconducting (SC)-ECR ion source and a deuterated polyethylene target, was developed at the Research Center for Nuclear Physics at Osaka University. A 3He2+ beam of 400 eµA was generated by the SC-ECR ion source with the acceleration voltage of 20 kV in an experiment that utilized the fusion reaction 3He + deuteron (D) → proton(P) + 4He. Protons with energies of the order of several MeV were successfully generated at 3.67 Hz at the atmosphere side of the target in the experimental setup, using a novel target base with a thin aluminum window.

Enhancement of 211At Uptake via the Sodium Iodide Symporter by the Addition of Ascorbic Acid in Targeted α-Therapy of Thyroid Cancer.

211At is an α-emitter that has similar chemical properties to iodine and is used in targeted α-therapy. In the present study, we added ascorbic acid (AA) to 211At solution to increase the radiochemical purity of astatide and evaluated its efficacy against differentiated thyroid cancer, which is characterized by the expression of sodium/iodide symporter (NIS). Methods: Crude 211At solution (AA(-)) and 211At solution treated with AA (AA(+)) were prepared. Uptake by the thyroid was compared between the 2 solutions in normal male Wistar rats (n = 6). Cellular uptake in K1-NIS cells was analyzed under the AA(+) and AA(-) conditions. AA(+) was injected at 3 doses into K1-NIS xenograft mice: 1 MBq (n = 6), 0.4 MBq (n = 6), and 0.1 MBq (n = 6), and vehicle was injected into control mice (n = 6). The treatment effects were compared among the 4 groups. Results: Uptake by the thyroid was significantly enhanced in rats injected with the AA(+) as compared with those injected with AA(-). Cellular uptake analysis showed significantly increased uptake of 211At by the K1-NIS cells under the AA(+) condition as compared with the AA(-) condition. In the mouse xenograft model, the K1-NIS tumors showed significant accumulation of 211At at 3 and 24 h after administration (22.5 ± 10.4 and 12.9 ± 6.8 percentage injected dose, respectively). Tumor growth was immediately inhibited in a dose-dependent manner after administration of 211At. In the survival analysis, the 211At groups (0.1, 0.4, and 1 MBq) showed significantly better survival than the control group. Conclusion: Uptake of 211At was enhanced in differentiated thyroid cancer cells as well as the normal thyroid using 211At solution treated with AA. The method also showed dose-dependent efficacy against the K1-NIS xenografts, suggesting its potential applicability to targeted α-therapy.

Foundations of layer-specific fMRI and investigations of neurophysiological activity in the laminarized neocortex and olfactory bulb of animal models.

Laminar organization of neuronal circuits is a recurring feature of how the brain processes information. For instance, different layers compartmentalize different cell types, synaptic activities, and have unique intrinsic and extrinsic connections that serve as units for specialized signal processing. Functional MRI is an invaluable tool to investigate laminar processing in the in vivo human brain, but it measures neuronal activity indirectly by way of the hemodynamic response. Therefore, the accuracy of high-resolution laminar fMRI depends on how precisely it can measure localized microvascular changes nearest to the site of evoked activity. To determine the specificity of fMRI responses to the true neurophysiological responses across layers, the flexibility to invasive procedures in animal models has been necessary. In this review, we will examine different fMRI contrasts and their appropriate uses for layer-specific fMRI, and how localized laminar processing was examined in the neocortex and olfactory bulb. Through collective efforts, it was determined that microvessels, including capillaries, are regulated within single layers and that several endogenous and contrast-enhanced fMRI contrast mechanisms can separate these neural-specific vascular changes from the nonspecific, especially cerebral blood volume-weighted fMRI with intravenous contrast agent injection. We will also propose some open questions that are relevant for the successful implementation of layer-specific fMRI and its potential future directions to study laminar processing when combined with optogenetics.

Dominance of layer-specific microvessel dilation in contrast-enhanced high-resolution fMRI: Comparison between hemodynamic spread and vascular architecture with CLARITY.

Contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI response peaks are specific to the layer of evoked synaptic activity (Poplawsky et al., 2015), but the spatial resolution limit of CBVw fMRI is unknown. In this study, we measured the laminar spread of the CBVw fMRI evoked response in the external plexiform layer (EPL, 265 ± 65 µm anatomical thickness, mean ± SD, n = 30 locations from 5 rats) of the rat olfactory bulb during electrical stimulation of the lateral olfactory tract and examined its potential vascular source. First, we obtained the evoked CBVw fMRI responses with a 55 × 55 µm2 in-plane resolution and a 500-µm thickness at 9.4 T, and found that the fMRI signal peaked predominantly in the inner half of EPL (136 ± 54 µm anatomical thickness). The mean full-width at half-maximum of these fMRI peaks was 347 ± 102 µm and the functional spread was approximately 100 or 200 µm when the effects of the laminar thicknesses of EPL or inner EPL were removed, respectively. Second, we visualized the vascular architecture of EPL from a different rat using a Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel (CLARITY)-based tissue preparation method and confocal microscopy. Microvascular segments with an outer diameter of <11 µm accounted for 64.3% of the total vascular volume within EPL and had a mean segment length of 55 ± 40 µm (n = 472). Additionally, vessels that crossed the EPL border had a mean segment length outside of EPL equal to 73 ± 61 µm (n = 28), which is comparable to half of the functional spread (50-100 µm). Therefore, we conclude that dilation of these microvessels, including capillaries, likely dominate the CBVw fMRI response and that the biological limit of the fMRI spatial resolution is approximately the average length of 1-2 microvessel segments, which may be sufficient for examining sublaminar circuits.

Inhibitory Neuron Activity Contributions to Hemodynamic Responses and Metabolic Load Examined Using an Inhibitory Optogenetic Mouse Model.

Hemodynamic signals are routinely used to noninvasively assess brain function in humans and animals. This work examined the contribution of inhibitory neuron activity on hemodynamic responses captured by changes in blood flow, volume and oxygenation in the cortex of lightly anesthetized mice. Because cortical activity is not commonly initiated by inhibitory neurons, experiments were conducted to examine the neuronal activity properties elicited by photo-stimulation. We observed comparable increases in neuronal activity evoked by forelimb and photo-stimulation; however, significantly larger increases in blood flow and volume were produced by photo-stimulation of inhibitory neurons compared with forelimb stimulation. Following blockade of glutamate and GABA-A receptors to reduce postsynaptic activity contributions, neuronal activity was reliably modulated and hemodynamic changes persisted, though slightly reduced. More importantly, photo-stimulation-evoked changes in blood flow and volume were suppressed by 75-80% with the administration of a nitric oxide synthase inhibitor, suggesting that inhibitory neurons regulate blood flow mostly via nitric oxide. Lastly, forelimb and photo-stimulation of excitatory neurons produced local decreases in blood oxygenation, while large increases were generated by photo-stimulation of inhibitory neurons. Estimates of oxygen metabolism suggest that inhibitory neuron activity has a small impact on tissue metabolic load, indicating a mismatch between the metabolic demand and blood flow regulation properties of inhibitory and excitatory neurons.

Optogenetic investigation of the variable neurovascular coupling along the interhemispheric circuits.

The interhemispheric circuit connecting the left and the right mammalian brain plays a key role in integration of signals from the left and the right side of the body. The information transfer is carried out by modulation of simultaneous excitation and inhibition. Hemodynamic studies of this circuit are inconsistent since little is known about neurovascular coupling of mixed excitatory and inhibitory signals. We investigated the variability in hemodynamic responses driven by the interhemispheric circuit during optogenetic and somatosensory activation. We observed differences in the neurovascular response based on the stimulation site - cell bodies versus distal projections. In half of the experiments, optogenetic stimulation of the cell bodies evoked a predominant post-synaptic inhibition in the other hemisphere, accompanied by metabolic oxygen consumption without coupled functional hyperemia. When the same transcallosal stimulation resulted in predominant post-synaptic excitation, the hemodynamic response was biphasic, consisting of metabolic dip followed by functional hyperemia. Optogenetic suppression of the postsynaptic excitation abolished the coupled functional hyperemia. In contrast, light stimulation at distal projections evoked consistently a metabolic response. Our findings suggest that functional hyperemia requires signals originating from the cell body and the hemodynamic response variability appears to reflect the balance between the post-synaptic excitation and inhibition.

Physics of epi-thermal boron neutron capture therapy (epi-thermal BNCT).

The physics of epi-thermal neutrons in the human body is discussed in the effort to clarify the nature of the unique radiologic properties of boron neutron capture therapy (BNCT). This discussion leads to the computational method of Monte Carlo simulation in BNCT. The method is discussed through two examples based on model phantoms. The physics is kept at an introductory level in the discussion in this tutorial review.

Functional Connectivity of Resting Hemodynamic Signals in Submillimeter Orientation Columns of the Visual Cortex.

Resting-state functional magnetic resonance imaging has been increasingly used for examining connectivity across brain regions. The spatial scale by which hemodynamic imaging can resolve functional connections at rest remains unknown. To examine this issue, deoxyhemoglobin-weighted intrinsic optical imaging data were acquired from the visual cortex of lightly anesthetized ferrets. The neural activity of orientation domains, which span a distance of 0.7-0.8 mm, has been shown to be correlated during evoked activity and at rest. We performed separate analyses to assess the degree to which the spatial and temporal characteristics of spontaneous hemodynamic signals depend on the known functional organization of orientation columns. As a control, artificial orientation column maps were generated. Spatially, resting hemodynamic patterns showed a higher spatial resemblance to iso-orientation maps than artificially generated maps. Temporally, a correlation analysis was used to establish whether iso-orientation domains are more correlated than orthogonal orientation domains. After accounting for a significant decrease in correlation as a function of distance, a small but significant temporal correlation between iso-orientation domains was found, which decreased with increasing difference in orientation preference. This dependence was abolished when using artificially synthetized orientation maps. Finally, the temporal correlation coefficient as a function of orientation difference at rest showed a correspondence with that calculated during visual stimulation suggesting that the strength of resting connectivity is related to the strength of the visual stimulation response. Our results suggest that temporal coherence of hemodynamic signals measured by optical imaging of intrinsic signals exists at a submillimeter columnar scale in resting state.

Improved spatial accuracy of functional maps in the rat olfactory bulb using supervised machine learning approach.

Functional MRI (fMRI) is a popular and important tool for noninvasive mapping of neural activity. As fMRI measures the hemodynamic response, the resulting activation maps do not perfectly reflect the underlying neural activity. The purpose of this work was to design a data-driven model to improve the spatial accuracy of fMRI maps in the rat olfactory bulb. This system is an ideal choice for this investigation since the bulb circuit is well characterized, allowing for an accurate definition of activity patterns in order to train the model. We generated models for both cerebral blood volume weighted (CBVw) and blood oxygen level dependent (BOLD) fMRI data. The results indicate that the spatial accuracy of the activation maps is either significantly improved or at worst not significantly different when using the learned models compared to a conventional general linear model approach, particularly for BOLD images and activity patterns involving deep layers of the bulb. Furthermore, the activation maps computed by CBVw and BOLD data show increased agreement when using the learned models, lending more confidence to their accuracy. The models presented here could have an immediate impact on studies of the olfactory bulb, but perhaps more importantly, demonstrate the potential for similar flexible, data-driven models to improve the quality of activation maps calculated using fMRI data.

Layer-Specific fMRI Responses to Excitatory and Inhibitory Neuronal Activities in the Olfactory Bulb.

High-resolution functional magnetic resonance imaging (fMRI) detects localized neuronal activity via the hemodynamic response, but it is unclear whether it accurately identifies neuronal activity specific to individual layers. To address this issue, we preferentially evoked neuronal activity in superficial, middle, and deep layers of the rat olfactory bulb: the glomerular layer by odor (5% amyl acetate), the external plexiform layer by electrical stimulation of the lateral olfactory tract (LOT), and the granule cell layer by electrical stimulation of the anterior commissure (AC), respectively. Electrophysiology, laser-Doppler flowmetry of cerebral blood flow (CBF), and blood oxygenation level-dependent (BOLD) and cerebral blood volume-weighted (CBV) fMRI at 9.4 T were performed independently. We found that excitation of inhibitory granule cells by stimulating LOT and AC decreased the spontaneous multi-unit activities of excitatory mitral cells and subsequently increased CBF, CBV, and BOLD signals. Odor stimulation also increased the hemodynamic responses. Furthermore, the greatest CBV fMRI responses were discretely separated into the same layers as the evoked neuronal activities for all three stimuli, whereas BOLD was poorly localized with some exception to the poststimulus undershoot. In addition, the temporal dynamics of the fMRI responses varied depending on the stimulation pathway, even within the same layer. These results indicate that the vasculature is regulated within individual layers and CBV fMRI has a higher fidelity to the evoked neuronal activity compared with BOLD. Our findings are significant for understanding the neuronal origin and spatial specificity of hemodynamic responses, especially for the interpretation of laminar-resolution fMRI. SIGNIFICANCE STATEMENT: Functional magnetic resonance imaging (fMRI) is a noninvasive, in vivo technique widely used to map function of the entire brain, including deep structures, in animals and humans. However, it measures neuronal activity indirectly by way of the vascular response. It is currently unclear how finely the hemodynamic response is regulated within single cortical layers and whether increased inhibitory neuronal activities affect fMRI signal changes. Both laminar specificity and the neural origins of fMRI are important to interpret functional maps properly, which we investigated by activating discrete rat olfactory bulb circuits.