Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Transcranial Focused Ultrasound to the Right Prefrontal Cortex Improves Mood and Alters Functional Connectivity in Humans.

Transcranial focused ultrasound (tFUS) is an emerging method for non-invasive neuromodulation akin to transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS). tFUS offers several advantages over electromagnetic methods including high spatial resolution and the ability to reach deep brain targets. Here we describe two experiments assessing whether tFUS could modulate mood in healthy human volunteers by targeting the right inferior frontal gyrus (rIFG), an area implicated in mood and emotional regulation. In a randomized, placebo-controlled, double-blind study, participants received 30 s of 500 kHz tFUS or a placebo control. Visual Analog Mood Scales (VAMS) assessed mood four times within an hour (baseline and three times after tFUS). Participants who received tFUS reported an overall increase in Global Affect (GA), an aggregate score from the VAMS scale, indicating a positive shift in mood. Experiment 2 examined resting-state functional (FC) connectivity using functional magnetic resonance imaging (fMRI) following 2 min of 500 kHz tFUS at the rIFG. As in Experiment 1, tFUS enhanced self-reported mood states and also decreased FC in resting state networks related to emotion and mood regulation. These results suggest that tFUS can be used to modulate mood and emotional regulation networks in the prefrontal cortex.

Ultrasonic Neuromodulation Causes Widespread Cortical Activation via an Indirect Auditory Mechanism.

Ultrasound has received widespread attention as an emerging technology for targeted, non-invasive neuromodulation based on its ability to evoke electrophysiological and motor responses in animals. However, little is known about the spatiotemporal pattern of ultrasound-induced brain activity that could drive these responses. Here, we address this question by combining focused ultrasound with wide-field optical imaging of calcium signals in transgenic mice. Surprisingly, we find cortical activity patterns consistent with indirect activation of auditory pathways rather than direct neuromodulation at the ultrasound focus. Ultrasound-induced activity is similar to that evoked by audible sound. Furthermore, both ultrasound and audible sound elicit motor responses consistent with a startle reflex, with both responses reduced by chemical deafening. These findings reveal an indirect auditory mechanism for ultrasound-induced cortical activity and movement requiring careful consideration in future development of ultrasonic neuromodulation as a tool in neuroscience research.

Augmented Reality Marker Hiding with Texture Deformation.

Augmented reality (AR) marker hiding is a technique to visually remove AR markers in a real-time video stream. A conventional approach transforms a background image with a homography matrix calculated on the basis of a camera pose and overlays the transformed image on an AR marker region in a real-time frame, assuming that the AR marker is on a planar surface. However, this approach may cause discontinuities in textures around the boundary between the marker and its surrounding area when the planar surface assumption is not satisfied. This paper proposes a method for AR marker hiding without discontinuities around texture boundaries even under nonplanar background geometry without measuring it. For doing this, our method estimates the dense motion in the marker's background by analyzing the motion of sparse feature points around it, together with a smooth motion assumption, and deforms the background image according to it. Our experiments demonstrate the effectiveness of the proposed method in various environments with different background geometries and textures.

Diminished Reality Based on Image Inpainting Considering Background Geometry.

Diminished reality aims to remove real objects from video images and fill in the missing regions with plausible background textures in real time. Most conventional methods based on image inpainting achieve diminished reality by assuming that the background around a target object is almost planar. This paper proposes a new diminished reality method that considers background geometries with less constraints than the conventional ones. In this study, we approximate the background geometry by combining local planes, and improve the quality of image inpainting by correcting the perspective distortion of texture and limiting the search area for finding similar textures as exemplars. The temporal coherence of texture is preserved using the geometries and camera pose estimated by visual-simultaneous localization and mapping (SLAM). The mask region that includes a target object is robustly set in each frame by projecting a 3D region, rather than tracking the object in 2D image space. The effectiveness of the proposed method is successfully demonstrated using several experimental environments.

Transcranial focused ultrasound modulates intrinsic and evoked EEG dynamics.

BACKGROUND: The integration of EEG recordings and transcranial neuromodulation has provided a useful construct for noninvasively investigating the modification of human brain circuit activity. Recent evidence has demonstrated that focused ultrasound can be targeted through the human skull to affect the amplitude of somatosensory evoked potentials and its associated spectral content. OBJECTIVE/HYPOTHESIS: The present study tests whether focused ultrasound transmitted through the human skull and targeted to somatosensory cortex can affect the phase and phase rate of cortical oscillatory dynamics. METHODS: A computational model was developed to gain insight regarding the insertion behavior of ultrasound induced pressure waves in the human head. The instantaneous phase and phase rate of EEG recordings before, during, and after transmission of transcranial focused ultrasound (tFUS) to human somatosensory cortex were examined to explore its effects on phase dynamics. RESULTS: Computational modeling results show the skull effectively reinforces the focusing of tFUS due to curvature of material interfaces. Neurophysiological recordings show that tFUS alters the phase distribution of intrinsic brain activity for beta frequencies, but not gamma. This modulation was accompanied by a change in phase rate of both beta and gamma frequencies. Additionally, tFUS modulated phase distributions in the beta band of early sensory-evoked activity but did not affect late sensory-evoked activity, lending support to the spatial specificity of tFUS for neuromodulation. This spatial specificity was confirmed through an additional experiment where the ultrasound transducer was moved 1 cm laterally from the original cortical target. CONCLUSIONS: Focused ultrasonic energy can alter EEG oscillatory dynamics through local mechanical perturbation of discrete cortical circuits.

Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans.

Improved methods of noninvasively modulating human brain function are needed. Here we probed the influence of transcranial focused ultrasound (tFUS) targeted to the human primary somatosensory cortex (S1) on sensory-evoked brain activity and sensory discrimination abilities. The lateral and axial spatial resolution of the tFUS beam implemented were 4.9 mm and 18 mm, respectively. Electroencephalographic recordings showed that tFUS significantly attenuated the amplitudes of somatosensory evoked potentials elicited by median nerve stimulation. We also found that tFUS significantly modulated the spectral content of sensory-evoked brain oscillations. The changes produced by tFUS on sensory-evoked brain activity were abolished when the acoustic beam was focused 1 cm anterior or posterior to S1. Behavioral investigations showed that tFUS targeted to S1 enhanced performance on sensory discrimination tasks without affecting task attention or response bias. We conclude that tFUS can be used to focally modulate human cortical function.

Distinct spatiotemporal activity in principal neurons of the mouse olfactory bulb in anesthetized and awake states.

The acquisition of olfactory information and its early processing in mammals are modulated by brain states through sniffing behavior and neural feedback. We imaged the spatiotemporal pattern of odor-evoked activity in a population of output neurons (mitral/tufted cells, MTCs) in the olfactory bulb (OB) of head-restrained mice expressing a genetically-encoded calcium indicator. The temporal dynamics of MTC population activity were relatively simple in anesthetized animals, but were highly variable in awake animals. However, the apparently irregular activity in awake animals could be predicted well using sniff timing measured externally, or inferred through fluctuations in the global responses of MTC population even without explicit knowledge of sniff times. The overall spatial pattern of activity was conserved across states, but odor responses had a diffuse spatial component in anesthetized mice that was less prominent during wakefulness. Multi-photon microscopy indicated that MTC lateral dendrites were the likely source of spatially disperse responses in the anesthetized animal. Our data demonstrate that the temporal and spatial dynamics of MTCs can be significantly modulated by behavioral state, and that the ensemble activity of MTCs can provide information about sniff timing to downstream circuits to help decode odor responses.

Pulsed ultrasound differentially stimulates somatosensory circuits in humans as indicated by EEG and FMRI.

Peripheral somatosensory circuits are known to respond to diverse stimulus modalities. The energy modalities capable of eliciting somatosensory responses traditionally belong to mechanical, thermal, electromagnetic, and photonic domains. Ultrasound (US) applied to the periphery has also been reported to evoke diverse somatosensations. These observations however have been based primarily on subjective reports and lack neurophysiological descriptions. To investigate the effects of peripherally applied US on human somatosensory brain circuit activity we recorded evoked potentials using electroencephalography and conducted functional magnetic resonance imaging of blood oxygen level-dependent (BOLD) responses to fingertip stimulation with pulsed US. We found a pulsed US waveform designed to elicit a mild vibration sensation reliably triggered evoked potentials having distinct waveform morphologies including a large double-peaked vertex potential. Fingertip stimulation with this pulsed US waveform also led to the appearance of BOLD signals in brain regions responsible for somatosensory discrimination including the primary somatosensory cortex and parietal operculum, as well as brain regions involved in hierarchical somatosensory processing, such as the insula, anterior middle cingulate cortex, and supramarginal gyrus. By changing the energy profile of the pulsed US stimulus waveform we observed pulsed US can differentially activate somatosensory circuits and alter subjective reports that are concomitant with changes in evoked potential morphology and BOLD response patterns. Based on these observations we conclude pulsed US can functionally stimulate different somatosensory fibers and receptors, which may permit new approaches to the study and diagnosis of peripheral nerve injury, dysfunction, and disease.

Light-emitting diodes for biological microscopy.

Various technological advances have made imaging an increasingly useful tool in the life sciences. Imaging techniques that move away from the limitations of wide-field microscopy have allowed deeper, higher-resolution imaging of thick biological tissue. Even within wide-field microscopy, advancements such as structured and sheet illumination, as well as improvements of biological probes, have led to better visualization of cells and their subcellular structures. The illumination source for wide-field microscopy, however, has remained relatively unchanged for decades, relying mainly on xenon, mercury, and halogen lamps. Light-emitting diodes (LEDs) have existed for more than 80 years, but their spectral range and output light flux have only recently become large enough for use in biological applications. This article presents the basic information necessary to build and to use an LED-based illumination source for microscopy. It also provides some useful resources about LED advancements. Although commercial LED-based illuminators for microscopy are available, custom-built illumination systems can incorporate the latest LED chips into microscopes more quickly and inexpensively than is possible through the retail market.

Optophysiological analysis of associational circuits in the olfactory cortex.

Primary olfactory cortical areas receive direct input from the olfactory bulb, but also have extensive associational connections that have been mainly studied with classical anatomical methods. Here, we shed light on the functional properties of associational connections in the anterior and posterior piriform cortices (aPC and pPC) using optophysiological methods. We found that the aPC receives dense functional connections from the anterior olfactory nucleus (AON), a major hub in olfactory cortical circuits. The local recurrent connectivity within the aPC, long invoked in cortical autoassociative models, is sparse and weak. By contrast, the pPC receives negligible input from the AON, but has dense connections from the aPC as well as more local recurrent connections than the aPC. Finally, there are negligible functional connections from the pPC to aPC. Our study provides a circuit basis for a more sensory role for the aPC in odor processing and an associative role for the pPC.

Human periosteum-derived cells combined with superporous hydroxyapatite blocks used as an osteogenic bone substitute for periodontal regenerative therapy: an animal implantation study using nude mice.

BACKGROUND: A superporous (85%) hydroxyapatite (HA) block was recently developed to improve osteoconductivity, but it was often not clinically successful when used to treat periodontal osseous defects. The primary purpose of this study is to develop a clinically applicable tissue-engineered bone substitute using this HA block and human alveolar periosteum-derived cells. METHODS: Commercially available superporous HA blocks were acid treated and subjected to a three-dimensional (3D) culture for periosteal cell cultivation. Cells in the pore regions of the treated HA block were observed on the fracture surface by scanning electron microscopy. After osteogenic induction, the cell-HA complexes were implanted subcutaneously in nude mice. Osteoid formation was histologically evaluated. RESULTS: Acid treatment enlarged the interconnections among pores, resulting in the deep penetration of periosteal cells. Under these conditions, cells were maintained for >2 weeks without appreciable cell death in the deep pore regions of the HA block. The cell-HA complexes that received in vitro osteogenic induction formed osteoids in pore regions of the treated HA blocks in vivo. In contrast, most pore regions in the non-pretreated, cell-free HA blocks that were evaluated in vivo remained cell free. CONCLUSIONS: Our findings suggest that an acid-treated HA block could function as a better scaffold for the 3D high-density culture of human periosteal cells in vitro, and this cell-HA complex had significant osteogenic potential at the site of implantation in vivo. Compared with the cell-free HA block, our cell-HA complex using periosteal cells, which are the most accessible for clinical periodontists, showed promising results as a bone substitute in periodontal regenerative therapy.

Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways.

Functional neuroimaging uses activity-dependent changes in cerebral blood flow to map brain activity, but the contributions of presynaptic and postsynaptic activity are incompletely understood, as are the underlying cellular pathways. Using intravital multiphoton microscopy, we measured presynaptic activity, postsynaptic neuronal and astrocytic calcium responses, and erythrocyte velocity and flux in olfactory glomeruli during odor stimulation in mice. Odor-evoked functional hyperemia in glomerular capillaries was highly correlated with glutamate release, but did not require local postsynaptic activity. Odor stimulation induced calcium transients in astrocyte endfeet and an associated dilation of upstream arterioles. Calcium elevations in astrocytes and functional hyperemia depended on astrocytic metabotropic glutamate receptor 5 and cyclooxygenase activation. Astrocytic glutamate transporters also contributed to functional hyperemia through mechanisms independent of calcium rises and cyclooxygenase activation. These local pathways initiated by glutamate account for a large part of the coupling between synaptic activity and functional hyperemia in the olfactory bulb.

LED arrays as cost effective and efficient light sources for widefield microscopy.

New developments in fluorophores as well as in detection methods have fueled the rapid growth of optical imaging in the life sciences. Commercial widefield microscopes generally use arc lamps, excitation/emission filters and shutters for fluorescence imaging. These components can be expensive, difficult to maintain and preclude stable illumination. Here, we describe methods to construct inexpensive and easy-to-use light sources for optical microscopy using light-emitting diodes (LEDs). We also provide examples of its applicability to biological fluorescence imaging.

Molecular characterization and expression of the cholesteryl ester transfer protein gene in chickens.

The cDNA for cholesteryl ester transfer protein (CETP), a protein that catalyzes cholesteryl ester transfer between very low density and high density lipoproteins in plasma, was isolated from chicken liver. When the recombinant protein was overexpressed in HEK293 cells, cholesteryl ester transfer activity was observed in media and cell lysates. By Northern blot analysis, chicken CETP mRNA expression was detected in liver, brain, heart, and spleen. Changes in chicken CETP mRNA expression and plasma CETP activity with nutritional state were examined and found to increase following dietary supplementation with cholesterol in a similar way as in humans. Both the hepatic CETP mRNA levels and plasma CETP activity were significantly lower in mature (i.e egg-laying) hens than in immature female chickens, but were unaffected by age in male animals. Similar changes to those observed in female chickens were observed upon estradiol administration of males. The present study is the first to report the molecular characterization of an avian CETP, and the impairments of CETP gene and activity, which might be regulated by estrogen, play an important role in egg production in laying hens, demonstrating species-specific differences in the lipid metabolism of avian and mammalian species.