Multi-sensory Gamma Stimulation Ameliorates Alzheimer's-Associated Pathology and Improves Cognition.

We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function.

Noninvasive Deep Brain Stimulation via Temporally Interfering Electric Fields.

We report a noninvasive strategy for electrically stimulating neurons at depth. By delivering to the brain multiple electric fields at frequencies too high to recruit neural firing, but which differ by a frequency within the dynamic range of neural firing, we can electrically stimulate neurons throughout a region where interference between the multiple fields results in a prominent electric field envelope modulated at the difference frequency. We validated this temporal interference (TI) concept via modeling and physics experiments, and verified that neurons in the living mouse brain could follow the electric field envelope. We demonstrate the utility of TI stimulation by stimulating neurons in the hippocampus of living mice without recruiting neurons of the overlying cortex. Finally, we show that by altering the currents delivered to a set of immobile electrodes, we can steerably evoke different motor patterns in living mice.

Iterative expansion microscopy.

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Publisher Correction: A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes in Neuroimaging.

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in the mammalian brain and can be readily applied to neuroimaging.

Vibrotactile stimulation at gamma frequency mitigates pathology related to neurodegeneration and improves motor function.

The risk for neurodegenerative diseases increases with aging, with various pathological conditions and functional deficits accompanying these diseases. We have previously demonstrated that non-invasive visual stimulation using 40 Hz light flicker ameliorated pathology and modified cognitive function in mouse models of neurodegeneration, but whether 40 Hz stimulation using another sensory modality can impact neurodegeneration and motor function has not been studied. Here, we show that whole-body vibrotactile stimulation at 40 Hz leads to increased neural activity in the primary somatosensory cortex (SSp) and primary motor cortex (MOp). In two different mouse models of neurodegeneration, Tau P301S and CK-p25 mice, daily exposure to 40 Hz vibrotactile stimulation across multiple weeks also led to decreased brain pathology in SSp and MOp. Furthermore, both Tau P301S and CK-p25 mice showed improved motor performance after multiple weeks of daily 40 Hz vibrotactile stimulation. Vibrotactile stimulation at 40 Hz may thus be considered as a promising therapeutic strategy for neurodegenerative diseases with motor deficits.

Gamma frequency sensory stimulation in mild probable Alzheimer's dementia patients: Results of feasibility and pilot studies.

Non-invasive Gamma ENtrainment Using Sensory stimulation (GENUS) at 40Hz reduces Alzheimer's disease (AD) pathology such as amyloid and tau levels, prevents cerebral atrophy, and improves behavioral testing performance in mouse models of AD. Here, we report data from (1) a Phase 1 feasibility study (NCT04042922, ClinicalTrials.gov) in cognitively normal volunteers (n = 25), patients with mild AD dementia (n = 16), and patients with epilepsy who underwent intracranial electrode monitoring (n = 2) to assess safety and feasibility of a single brief GENUS session to induce entrainment and (2) a single-blinded, randomized, placebo-controlled Phase 2A pilot study (NCT04055376) in patients with mild probable AD dementia (n = 15) to assess safety, compliance, entrainment, and exploratory clinical outcomes after chronic daily 40Hz sensory stimulation for 3 months. Our Phase 1 study showed that 40Hz GENUS was safe and effectively induced entrainment in both cortical regions and other cortical and subcortical structures such as the hippocampus, amygdala, insula, and gyrus rectus. Our Phase 2A study demonstrated that chronic daily 40Hz light and sound GENUS was well-tolerated and that compliance was equally high in both the control and active groups, with participants equally inaccurate in guessing their group assignments prior to unblinding. Electroencephalography recordings show that our 40Hz GENUS device safely and effectively induced 40Hz entrainment in participants with mild AD dementia. After 3 months of daily stimulation, the group receiving 40Hz stimulation showed (i) lesser ventricular dilation and hippocampal atrophy, (ii) increased functional connectivity in the default mode network as well as with the medial visual network, (iii) better performance on the face-name association delayed recall test, and (iv) improved measures of daily activity rhythmicity compared to the control group. These results support further evaluation of GENUS in a pivotal clinical trial to evaluate its potential as a novel disease-modifying therapeutic for patients with AD.

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems.

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.

Fluidic measurement of electric field sensitivity of Ti-GaAs Schottky junction gated field effect biosensors.

We report the electric field and pH sensitivity of fluid gated metal-semiconductor hybrid (MSH) Schottky structures consisting of a Titanium layer on n-type GaAs. Compared to standard field-effect sensors, the MSH Schottky structures are 21 times more sensitive to electric field of -46.6 V/cm and show about six times larger resistance change as pH of the solution is decreased from 8.17 to 5.54. The potential change at the fluidic gate and passivation layer interface by bias voltages and pH are mirrored by the metal shunt, resulting in larger depletion widths under the Schottky junction and resistance change as compared to sensors with no Schottky junction. 2D numerical simulation results are in good agreement with the measured data and suggest thinner mesa with lower doping density can further increase device sensitivity.

Dielectrophoresis-based cell manipulation using electrodes on a reusable printed circuit board.

Particle manipulation based on dielectrophoresis (DEP) can be a versatile and useful tool in lab-on-chip systems for a wide range of cell patterning and tissue engineering applications. Even though there are extensive reports on the use of DEP for cell patterning applications, the development of approaches that make DEP even more affordable and common place is still desirable. In this study, we present the use of interdigitated electrodes on a printed circuit board (PCB) that can be reused to manipulate and position HeLa cells and polystyrene particles over 100 microm thick glass cover slips using DEP. An open-well or a closed microfluidic channel, both made of PDMS, was placed on the glass coverslip, which was then placed directly over the PCB. An AC voltage was applied to the electrodes on the PCB to induce DEP on the particles through the thin glass coverslip. The HeLa cells patterned with DEP were subsequently grown to confirm the lack of any adverse affects from the electric fields. This alternative and reusable platform for DEP particle manipulation can provide a convenient and rapid method for prototyping a DEP-based lab-on-chip system, cost-sensitive lab-on-chip applications, and a wide range of tissue engineering applications.

Advances in the automation of whole-cell patch clamp technology.

Electrophysiology is the study of neural activity in the form of local field potentials, current flow through ion channels, calcium spikes, back propagating action potentials and somatic action potentials, all measurable on a millisecond timescale. Despite great progress in imaging technologies and sensor proteins, none of the currently available tools allow imaging of neural activity on a millisecond timescale and beyond the first few hundreds of microns inside the brain. The patch clamp technique has been an invaluable tool since its inception several decades ago and has generated a wealth of knowledge about the nature of voltage- and ligand-gated ion channels, sub-threshold and supra-threshold activity, and characteristics of action potentials related to higher order functions. Many techniques that evolve to be standardized tools in the biological sciences go through a period of transformation in which they become, at least to some degree, automated, in order to improve reproducibility, throughput and standardization. The patch clamp technique is currently undergoing this transition, and in this review, we will discuss various aspects of this transition, covering advances in automated patch clamp technology both in vitro and in vivo.

Gamma Entrainment Binds Higher-Order Brain Regions and Offers Neuroprotection.

Neuronal and synaptic loss is characteristic in many neurodegenerative diseases, such as frontotemporal dementia and Alzheimer's disease. Recently, we showed that inducing gamma oscillations with visual stimulation (gamma entrainment using sensory stimuli, or GENUS) reduced amyloid plaques and phosphorylated tau in multiple mouse models. Whether GENUS can affect neurodegeneration or cognitive performance remains unknown. Here, we demonstrate that GENUS can entrain gamma oscillations in the visual cortex, hippocampus, and prefrontal cortex in Tau P301S and CK-p25 mouse models of neurodegeneration. Tau P301S and CK-p25 mice subjected to chronic, daily GENUS from the early stages of neurodegeneration showed a preservation of neuronal and synaptic density across multiple brain areas and modified cognitive performance. Our transcriptomic and phosphoproteomic data suggest that chronic GENUS shifts neurons to a less degenerative state, improving synaptic function, enhancing neuroprotective factors, and reducing DNA damage in neurons while also reducing inflammatory response in microglia.

Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo.

Targeted patch-clamp recording is a powerful method for characterizing visually identified cells in intact neural circuits, but it requires skill to perform. We previously developed an algorithm that automates "blind" patching in vivo, but full automation of visually guided, targeted in vivo patching has not been demonstrated, with currently available approaches requiring human intervention to compensate for cell movement as a patch pipette approaches a targeted neuron. Here we present a closed-loop real-time imaging strategy that automatically compensates for cell movement by tracking cell position and adjusting pipette motion while approaching a target. We demonstrate our system's ability to adaptively patch, under continuous two-photon imaging and real-time analysis, fluorophore-expressing neurons of multiple types in the living mouse cortex, without human intervention, with yields comparable to skilled human experimenters. Our "imagepatching" robot is easy to implement and will help enable scalable characterization of identified cell types in intact neural circuits.

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.

A three-line lateral flow assay strip for the measurement of C-reactive protein covering a broad physiological concentration range in human sera.

The lateral flow assay (LFA) strip sensor possesses many advantages as a diagnostic device, including the capabilities of rapid, one-step assay performance, and high throughput production. A major limitation of the sensor, however, is its difficulty in measuring a broad concentration range of target proteins, including C-reactive protein (CRP), due to the "hook effect." In this study, we report the use of a three-line LFA strip sensor, adding an antigen line to the conventional two-line LFA sensor, for detecting CRP within a broad concentration range in human sera. We introduced an antigen line between test and control lines in the LFA sensor. The antigen line was formed by dispensing a CRP antibody solution followed by a CRP solution in nitrocellulose membrane. All other conditions were identical to those applied to the conventional LFA strip sensor. The CRP level in test samples was generated by data processing from the intensities of three lines. The strip sensor measured a linear detection range of CRP concentration from 1 ng/mL to 500 µg/mL within 10 min, with a calculated detection range of 0.69 ng/mL-1.02 mg/mL. Using the developed three-line LFA sensor, 50 clinical samples were measured at a detection range of 0.4-84.7 µg/mL. This novel and easy-to-use CRP sensor can be a useful tool for rapid, sensitive, and cost-effective detection of a broad physiological concentration range of CRP capabilities that are vital for various diagnostic applications.

Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk.

We demonstrated the new antibody/gold nanoparticle/magnetic nanoparticle nanocomposites (antibody/AuNP/MNPs) and their application in the detection of the foodborne pathogen, Staphylococcus aureus (S. aureus), in milk. The nanocomposites were synthesized by coating the MNPs with bovine serum albumin (BSA) then adsorbing the AuNPs and anti-S. aureus antibodies on their surface. Using the completed immunomagnetic nanostructures, S. aureus inoculated in the milk sample was captured and isolated from the medium using the permanent magnet. The nanoparticle-bound cells as well as the unbound cells in the supernatant were enumerated via surface plating to evaluate the target binding capacity of the nanocomposites. The capture efficiencies of the antibody/AuNP/MNPs were 96% and 78% for S. aureus in PBS and the milk sample respectively, which were significantly higher than those of the antibody-coupled MNPs without any AuNP. The captured cells were also applied to the selective filtration system to produce color signals that were used for the detection of the target pathogen. During the filtration, the cells bound to the antibody/AuNP/MNPs remained on the surface of the membrane filter while unbound nanoparticles passed through the uniform pores of the membrane. After the gold enhancement, the cells-particles complex resting on the membrane surface rendered a visible color, and the signal intensity became higher as the target cell concentration increased. The detection limits of this colorimetric sensor were 1.5×10(3) and 1.5×10(5)CFU for S. aureus in PBS and the milk sample respectively. This sensing mechanism also had the high specificity for S. aureus over the other pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The assay required only 40min to obtain the results. With the use of the appropriate antibodies, our immunomagnetic nanocomposites-based detection strategy can provide an easy, convenient, and rapid sensing method for a wide range of pathogens.