The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Psilocybin has profound therapeutic potential for various mental health disorders, but its mechanisms of action are unknown. Functional MRI studies have reported the effects of psilocybin on brain activity and connectivity; however, these measurements rely on neurovascular coupling to infer neural activity changes and assume that blood flow responses to neural activity are not altered by psilocybin. Using two-photon excited fluorescence imaging in the visual cortex of awake mice to simultaneously measure neural activity and capillary blood flow dynamics, we found that psilocybin administration prolonged the increase in visual stimulus-evoked capillary blood flow - an effect which was reduced by pretreatment with a 5-HT2AR antagonist - despite not causing changes in the stimulus-evoked neural response. Multimodal widefield imaging also showed that psilocybin extends the stimulus-evoked vascular responses in surface vessels with no observed effect on the population neural response. Computational simulation with a whole-brain neural mass model showed that prolonged neurovascular coupling responses can lead to spurious increases in BOLD-based measures of functional connectivity. Together, these findings demonstrate that psilocybin broadens neurovascular responses in the brain and highlights the importance of accounting for these effects when interpreting human neuroimaging data of psychedelic drug action.

Alzheimer’s disease (AD) is characterized by progressive, irreversible neurodegeneration, leading to memory loss and cognitive decline. In mouse models of AD, global decreases in cerebral blood flow (CBF) are brought on by the plugging of capillaries by arrested neutrophils, and the administration of the neutrophil-specific antibody against Ly6G (anti-Ly6G) reduces these capillary stalls in minutes and improves cognitive function within hours. This suggests that at least some aspects of neural activity impairment are reversible, but the mechanism of this recovery – and what specific neural activity is normalized – is not yet known. In agreement with prior studies, we found orientation tuning selectivity to drifting gratings in primary visual cortex neurons to be broadened in mouse models of AD. Here, we hypothesized that the impaired neural response can be modified by blood flow improvement with anti-Ly6G treatment (4 mg/kg) in the APP/PS1 mouse model of AD. We transfected neurons in layer 2/3 of the primary visual cortex (V1) of mice with a fluorescent calcium indicator using AAV9 vectors (pAAV.Syn.GCaMP6s.WPRE.SV40, 10^12 vg/mL). We also injected the fluorescent labels methoxy-X04 to detect amyloid plaques and Texas-red-dextran in the vasculature to detect blocked capillaries. Drifting grating visual stimuli were presented to anesthetized mice with using MouseGoggles during recording with two-photon microscopy before and one day after anti-Ly6G or isotype control antibody administration (Fig. 1). One day after anti-Ly6G administration to reduce stalls and increase CBF, we observed a significant sharpening of orientation tuning relative to baseline in the same animals, as well as a trend toward increased spontaneous activity in V1 neurons (Fig. 2). Such normalization of neural activity patterns likely underlies the improved cognitive function that is seen within hours of blood flow increase in AD mouse models. Our data suggests some aspects of the neural and behavioral deficits in AD are acutely recoverable by increasing CBF. Such recovery demonstrates a promising avenue for future therapeutic targets to combat the symptoms of AD in humans.

Spatial disorientation is an early symptom of Alzheimer’s disease (AD). The hippocampus creates a cognitive map, wherein cells form firing fields in specific locations within an environment, termed place cells. Critically, place cells remain stable across visits to an environment, but change their firing rate or field location in a different environment. In rodent models of AD-like pathology, place cells exhibit broadened tuning and altered responses to environmental changes. Additionally, events known as sharp wave ripples (SWRs), coordinate the firing of hippocampal neurons, including place cells, and are disrupted in mouse models of AD. Our lab found that increasing cerebral blood flow (CBF) with anti-Ly6G antibody treatment improved performance on spatial memory tasks. Here, we investigate the effect of increased CBF on neural mechanisms associated with cognitive map stability across contexts. We investigated the association between CBF and cognitive map stability in 7-9-month-old APP/PS1 mice and wild-type controls by recording neural activity in hippocampus area CA1 using 64-channel silicon probes. Mice were recorded as they explored open field arenas with differing environmental contexts, A and B. Place cells were identified as neurons with significant spatial information in their firing rate maps. Local field potential recordings from pyramidal and stratum radiatum layers of CA1 were used to detect awake-SWRs (aSWRs). Place cells from APP/PS1 mice did not exhibit higher place field correlations within contexts (AA' or BB'), as compared to between (AB) contexts, suggesting these different contexts are not differentially encoded (Figure 1). In contrast, wild-type control mice showed higher within context correlation (Figure 1). APP/PS1 mice also had a reduced rate and duration of aSWR compared to controls (Figure 2). Following treatment with anti-Ly6G antibodies, both context discrimination by place cells and duration of aSWR increased in APP/PS1 mice (Figure 2). These results highlight the association between place cell stability and subsequent aSWR dynamics as a potential mechanism contributing to an impaired cognitive map in AD. Importantly, the rescue of behavioral features and aSWR physiological properties following anti-Ly6G antibody treatment suggests that increasing CBF may be a candidate therapy to mitigate spatial memory impairments in AD.

Neurodegenerative disease ultimately leads to altered neural activity patterns associated with impaired function. We examine these changes in mouse models of Alzheimer's disease, as well as their normalizaiton with increases in cerrebral blood flow. Full-text article not available; see video presentation

Transient stoppages of red blood cell (RBC) flow through capillaries—termed capillary stalls—occur persistently in neurological disorders such as Alzheimer’s disease and ischemic stroke and can interrupt oxygen delivery and exacerbate neurological damage. Effective imaging tools and analyses are necessary to understand the nature, role, and prevention of stalls. In this study, we dissect differences in stalls measured by two-photon Bessel beam microscopy (Bessel-2PM) and optical coherence tomography (OCT) to gain insight into the temporal dynamics of stalls. Twenty-minute series of volumetric angiograms were obtained separately with Bessel-2PM and OCT on the same day in awake, head-fixed mice. The temporal dynamics of stalling in both methods revealed a minority population of susceptible capillaries that exhibited frequent stalls and a large majority of capillaries with infrequent stalls. Differences between OCT and Bessel-2PM in the repeatability and dynamics of stalls are explained by differences in their sensitivity to short or infrequent stalls based on scanning speed and detection off-time. Finally, stroke caused a shift toward the frequently stalling capillary subpopulation, lasting 1 week post-stroke. Dynamic stall analysis therefore enables examination of physiological and methodological contributions to the stalls measured in disease models and across studies.

Myocardial blood flow deficits in heart failure with preserved ejection fraction (HFpEF) patients and related animal models have been recognized for decades, but the underlying mechanisms and resulting consequences for HFpEF pathogenesis remain poorly understood. Using intravital cardiac microcopy in a two-hit mouse model of HFpEF, we identified an increased number of neutrophils in capillaries slow moving orstalled, blocking blood flow, as compared to control mice. Administration of antibodies against the neutrophil marker Ly6G reduced the number of arrested neutrophils in myocardial capillaries, leading to both a reduction in myocardial tissue hypoxia and improvement in diastolic function and exercise tolerance. This study identifies a previously uncharacterized cellular mechanism that explains myocardial blood flow deficits in mouse models of HFpEF, and demonstrates that improving myocardial blood flow improves heart function. Restoring myocardial perfusion by preventing neutrophil arrest in coronary capillaries may provide a strategy for improving heart function in HFpEF patients. ### Competing Interest Statement The authors have declared no competing interest.

Several genetic and cardiovascular risk factors increase incidence of Alzheimer’s disease and related dementias (ADRD). Hypertension and the ε4 allele of apolipoprotein E (ApoE) are powerful drivers of cognitive impairment in ADRD. These risk factors are also associated with decreased cerebral blood flow (CBF). Experimental data suggest that the CBF reduction may be due to temporary capillary occlusions (capillary stalls) caused by circulating vascular and immune cells. To gain insight into how these risk factors may contribute to cognitive decline, we induced hypertension in mice expressing human ApoE3 or E4 (ApoE3- or E4-TR mice), and focused on the resulting CBF and cognitive alterations, and their potential reversal by preventing capillary stalling. Male ApoE3 and 4-TR mice (n = 9-15/group) were assessed at three timepoints: Baseline, following two weeks of angiotensin II-induced hypertension (AngII, 500ng/kg/min), and after an additional week of anti-platelet treatment (prasugrel, 10mg/kg, daily). We tested spatial memory function in a Y-maze, and imaged cortical microvascular flow using two-photon microscopy. At baseline, ApoE3 and 4-TR mice showed similar memory performance, but with hypertension (∼25 mmHg increase in blood pressure) ApoE4-TR mice exhibited a spatial working memory deficit, which coincided with a 26% decrease in capillary flow speed (4.11±0.3 vs. 3.05±0.29 mm/s, p <0.01), and a 1.8X increase in the incidence of non-flowing capillaries. Most stalled capillaries contained only red blood cells in the stalled segment (57%), with some also containing leukocytes (30%), or platelets (12%) (Fig. 1). Treatment with prasugrel led to a 55% reduction in stalling (65±9 vs. 144±24 stalls/mm 3 ), and a concomitant 23% increase in capillary blood flow (3.75±0.36 vs. 3.05±0.29 mm/s, p = 0.11), and significantly improved blood flow in penetrating arterioles that correlated with restored performance on the Y-maze task (Fig. 2). Immunohistochemistry further revealed improved BBB integrity and reduced gliosis following prasugrel treatment. Paralleling human findings, we show that mice with the e4 allele of ApoE experience more deleterious consequences from hypertension, as compared to their e3 counterparts. Prasugrel treatment led to an almost complete recovery of these deficits, suggesting that anti-platelet agents may be of therapeutic value by improving CBF and cognitive function in the presence of these risk factors.

Alzheimer’s disease (AD) manifests with early spatial memory impairment and is linked to the degeneration of hippocampal circuits. Hippocampal sharp wave ripples (SWRs) are high-frequency population-burst events that coordinate the reactivation of neural assemblies (groups of neurons that become correlated in their firing patterns during learning) in post-learning sleep, which is the neural basis of memory consolidation. SWRs are reduced in the APP/PS1 mouse model of AD-like pathology. Previously, we showed that cerebral blood flow (CBF) decreases and memory deficits were rescued following treatment with anti-Ly6G antibodies. Here, we examine the potential normalization of hippocampal circuit activity with CBF increase. Male, 7-14-month-old APP/PS1 mice and wild-type controls were implanted with 64-channel silicon probes in hippocampal area CA1. Neural activity was recorded during sleep before and after the exploration of an open field. Putative cell types were identified using feature-based classification, and neural assemblies were detected using independent component analysis. APP/PS1 mice had reduced magnitude and duration of assembly reactivation in post-task sleep SWRs. After treatment with anti-Ly6G antibodies, which increase CBF and improve memory performance, we found increased reactivation of these assemblies in post-task sleep SWRs, relative to no-treatment controls (Figure 1). We found that increasing CBF normalizes neural mechanisms of memory consolidation that are altered in AD mouse models, supporting the development of treatment approaches to increase CBF in AD.

The study objective was to examine how the changes in outdoor concentration of traffic-related aerosol at schools occurring due to an anti-idling campaign affect the indoor aerosol concentration. Four urban public schools featuring different average numbers of school buses and cars and located at different distances from major urban traffic arteries were selected in Cincinnati (Ohio, USA). For each school, the indoor and outdoor air monitoring was conducted before and after the implementation of anti-idling campaign. Additionally, the ambient air was monitored at four "community" sites representing the background aerosol. The PM2.5 samples were collected and subjected to gravimetric, carbon and elemental analysis. The outdoor concentrations of PM2.5 and its relevant elemental constituents were consistent with the data reported in previous studies. Following the anti-idling campaign, the outdoor concentration of most elements decreased at two schools although only a fraction showed statistically significant changes. After accounting for the background aerosol changes, the positive impact of the anti-idling campaign was demonstrated only for one school – with the most intense local traffic. For this school, reduction of the outdoor elemental concentrations was followed by a decrease in the corresponding indoor concentrations (R2 = 0.47, p < 0.05). The data suggests that in cases when traffic emission is the dominant pollution source in the school vicinity, the changes in outdoor air quality associated with the anti-idling campaign are capable of reducing the children exposure to traffic aerosols inside the schools.

Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact during imaging or other measurements. Anesthetized animals must be closely monitored for the safety of the animals and to prevent unintended effects of altered physiology on experimental outcomes. Many currently available monitoring devices are expensive, invasive, or interfere with experimental design. Here, we present MousePZT, a low-cost device based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate matches those of commercial systems. Using the widely-used gas isoflurane and injectable ketamine/xylazine combination, we also demonstrate that changes in respiratory rate are more easily detected and can precede changes in heart rate associated with variations in anesthetic depth. Additional circuitry on the device outputs a respiration-locked trigger signal for respiratory-gating of imaging or other data acquisition and has high sensitivity and specificity for detecting respiratory cycles. We provide detailed instruction documents and all necessary microcontroller and computer software, enabling straightforward construction and utilization of this device.

In partial onset epilepsy, seizures arise focally in the brain and often propagate, causing acute behavior changes, chronic cognitive decline, and increased mortality. Patients frequently become refractory to medical management, leaving neurosurgical resection of the seizure focus as a primary treatment, which can cause neurologic deficits. In the cortex, focal seizures are thought to spread through horizontal connections in layers II/III, suggesting that selectively severing these connections could block seizure propagation while preserving normal columnar circuitry and function. We induced focal neocortical epilepsy in mice and used tightly-focused femtosecond-duration laser pulses to create a sub-surface, opencylinder cut surrounding the seizure focus and severing cortical layers II-IV. We monitored seizure propagation using electrophysiological recordings at the seizure focus and at distant electrodes for 3-8 months. With laser cuts, only 5% of seizures propagated to the distant electrodes, compared to 85% in control animals. Laser cuts also decreased the number of seizures that were initiated, so that the average number of propagated seizures per day decreased from 42 in control mice to 1.5 with laser cuts. Physiologically, these cuts produced a modest decrease in cortical blood flow that recovered within days and, at one month, left a ~20-μm wide scar with increased gliosis and localized inflammatory cell infiltration but minimal collateral damage. When placed over motor cortex, cuts did not cause notable deficits in a skilled reaching task. Femtosecond laser produced sub-surface cuts hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy, as might develop following traumatic brain injury. Once sentence summary In a mouse model of focal epilepsy, sub-surface laser-produced cuts encircling the seizure focus attenuate propagation without behavioral impairment.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 μm into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 μm of the murine spinal cord, in vivo.

Here, we report a magnetogenetic system, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity when exposed to magnetic fields. Adeno-associated virus (AAV)-mediated delivery of a floxed nanobody-TRPV1 into the striatum of adenosine-2a receptor-Cre drivers resulted in motor freezing when placed in a magnetic resonance imaging machine or adjacent to a transcranial magnetic stimulation device. Functional imaging and fiber photometry confirmed activation in response to magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing Cre into the globus pallidus led to similar circuit specificity and motor responses. Last, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in the subthalamic nucleus in PitX2-Cre parkinsonian mice resulted in reduced c-fos expression and motor rotational behavior. These data demonstrate that magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits noninvasively in vivo using clinically available devices.

Small-animal virtual reality (VR) systems have become invaluable tools in neuroscience for studying complex behavior during head-fixed neural recording, but they lag behind commercial human VR systems in terms of miniaturization, immersivity and advanced features such as eye tracking. Here we present MouseGoggles, a miniature VR headset for head-fixed mice that delivers independent, binocular visual stimulation over a wide field of view while enabling eye tracking and pupillometry in VR. Neural recordings in the visual cortex validate the quality of image presentation, while hippocampal recordings, associative reward learning and innate fear responses to virtual looming stimuli demonstrate an immersive VR experience. Our open-source system's simplicity and compact size will enable the broader adoption of VR methods in neuroscience.

Third harmonic generation with 1320-nm, femtosecond pulses can visualize individual myelinated axons in subcortical white matter through intact cortex of live mice. In a cuprizone multiple sclerosis model, this enabled longitudinal tracking of myelin damage.

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triple-helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments.