Anna Devor

Introduction: Gamma entrainment using sensory stimuli (GENUS) uses 40 Hz-pulsed sensory stimuli to entrain neural activity in the gamma band (30–150 Hz). However, the effect of GENUS on low-frequency vascular oscillations has not been fully explored. Objectives: The objective of this study is to elucidate the effect of GENUS on vasomotion in healthy mice and potential confounds for future application in disease studies. Methods: Head-fixed, awake C57Bl/6 mice ( n = 18; 9M and 9F) aged between 18 and 60 weeks were subjected to white light of either 40 Hz visual flicker (GENUS), or constant stimulus (Control). Blood flow was imaged using laser speckle contrast imaging (LSCI) before, during, immediately after 1 h of stimulus, and 30 min after the stimulus termination. Results: A linear mixed-effects model showed that GENUS enhanced the magnitude of 0.2–0.4 Hz blood flow oscillations by 38% during stimulation and by 30% at 30 min after stimulation compared to Control when controlled for age, sex, and other factors. The effect on vasomotion was distributed across the surface of many cortical regions not limited to visual areas. Conclusion: These results support the exploration of GENUS for increasing vasomotion in therapeutic contexts.

Introduction Gamma entrainment using sensory stimuli (GENUS) uses 40Hz-pulsed sensory stimuli to entrain neural activity in the gamma band (30-150Hz). However, the effect of GENUS on low-frequency vascular oscillations has not been fully explored. Objectives The objective of this study is to elucidate the effect of GENUS on vasomotion in healthy mice and potential confounds for future application in disease studies. Methods Head-fixed, awake C57Bl/6 mice (n=18; 9M 9F) aged between 18 to 60 weeks were subjected to white light of either 40Hz visual flicker (GENUS), or constant stimulus (control). Blood flow was imaged using laser speckle contrast imaging (LSCI) before, during, immediately after 1 hour of stimulus, and 30min after the stimulus termination. Results A linear mixed-effects model showed that GENUS enhanced the magnitude of 0.2-0.4Hz blood flow oscillations by 38% during stimulation and by 30% at 30 minutes after stimulation compared to control when controlled for age, sex, and other factors. The effect on vasomotion was distributed across many cortical regions not limited to visual areas and lasted beyond 24 hours post-stimulus. Conclusion These results support the exploration of GENUS for increasing vasomotion in therapeutic contexts.

During forebrain development, inhibitory interneurons and oligodendrocyte progenitor cells migrate long distances into the developing dorsal cortex. Human induced pluripotent stem cell-derived forebrain assembloids (FAs) provide direct experimental access to this migratory process in vitro. Using viral labeling to express yellow fluorescent protein (EYFP) and tandem-dimer tomato (tdTomato) driven by EF1α or SOX10 promoters, respectively, we tracked cells in FAs over 15-17h using spinning disk confocal microscopy. We developed an end-to-end processing pipeline for 4D volumetric imaging data, consisting of background subtraction and drift correction, manual cell coordinate tracking, and an analysis workflow to describe migratory cell behavior. Image preprocessing significantly improved data quality for subsequent manual tracking in datasets with heterogeneous labeling density and brightness. Trajectory analysis of 336 EYFP- and 337 tdTomato-labeled cells from twelve FAs indicates that most cells show super-diffusive directed motility. Our pipeline represents a key resource for cell tracking in FAs and similar three-dimensional platforms. This pipeline represents the first open tracking resource for iPSC-derived FAs and can be used as a ground-truth resource for the development of automated cell detection and tracking algorithms.

This editorial explores the novel The Secret of Secrets by Dan Brown as a gateway to longstanding debates in neuroscience, particularly surrounding consciousness and free will. Highlighting the novels references to real neurophotonic technologies, it examines where scientific evidence ends and speculative fiction begins.

Neuromodulatory transmitters have vasoactive properties. Therefore, the impulse response function (IRF) linking spontaneous neuronal activity with hemodynamics may depend on neuromodulation. To test this hypothesis, we used optical imaging to measure norepinephrine (NE) or acetylcholine (ACh), calcium (Ca²⁺) activity of cortical neurons and hemodynamics in cerebral cortex in awake mice. We show that modeling of hemodynamics as a weighted sum of Ca²⁺-specific and NE-specific IRFs (IRF_Ca2+ and IRF_NE) convolved with the respective time courses dramatically improved the model performance compared to using IRF_Ca2+ alone. In contrast to NE, ACh was largely redundant with Ca²⁺ and, therefore, did not improve the hemodynamic estimation. Because NE covaried with arousal, we observed instances of the diminished hemodynamic coherence between cortical regions during high arousal despite coherent behavior of the underlying neuronal Ca²⁺ activity. We conclude that, without accounting for noradrenergic neuromodulation, diminished hemodynamic coherence can be falsely interpreted as neuronal desynchronizations in neuroimaging studies.

Human cortical organoids (hCOs) are three-dimensional neural cell aggregates that recapitulate certain structural and functional aspects of the developing human cortex. Xenotransplantation of hCOs into the rodent brain enables human-centric modeling of neurodevelopmental processes in a physiologically relevant environment. Here, we present a neurorecording toolkit for longitudinal structural and functional assessment of hCO xenografts as they mature in vivo. Single hCOs were implanted into the retrosplenial cortex of adult immunodeficient mice and monitored for up to 8 months. Optical coherence tomography was used for label-free imaging of xenograft vascularization and structure, enabling quantitative assessments of capillary density and graft volume. To probe neuronal activity, human neurons were labeled with a calcium sensor before implantation using either adeno-associated or lentivirus for sparse or dense neuronal labeling, respectively. Fluorescent imaging was conducted using two-photon, widefield, and swept confocally-aligned planar excitation microscopy for single cell, whole-graft, and volumetric calcium imaging, respectively. Results from these modalities indicate an increase in neuronal activity and synchronicity over time during in vivo graft maturation. Further, we chronically implanted surface graphene microelectrode arrays (gMEAs) and performed recordings of xenograft and host local field potential signals simultaneously with 2P calcium imaging, confirming the spatial localization and human origin of electrical signals recorded at the xenograft surface.

The editorial reflects on the secret ingredients that can elevate the flavor of our scientific ideas.

Cerebral blood flow relies on continuous perfusion of capillaries to distribute and deliver oxygen and other nutrients, with very little energy reserves to tolerate disruptions in flow.^1,2 Recently, such disruptions have not only been observed at the capillary level, but observed with increased frequency in models of Alzheimer’s disease³ and stroke,^4,5 and mild traumatic brain injury (mTBI).⁶ Both modeling and measurement have shown that these events are disruptive to cerebral blood flow at the network scale.^{7 – 9} However, the acute effects of a single stall in capillary flow have not been well investigated. As blood flow is stopped for the duration of the stall, there is potential for acute hypoxia, although this has not been directly measured aside from anecdotally.^5,10 However, a recent study using a bioluminescent oxygen indicator associated hypoxic pockets with the nearby presence of injected microspheres,¹¹ offering strong evidence of stall-associated hypoxia. As stalls can have durations that are minutes long,^3,5,12 and occur repeatedly in the same capillaries, there is the potential for extended and repeated exposure to low levels of oxygen. Further, heterogeneous flow and vascular density in the microvascular network result in cortical tissue oxygen also being highly variable,¹³ meaning stalls could have differing impacts depending on their location in the network. Together, this raises the question of to what degree individual stalls acutely affect oxygen delivery in the brain and if this could be a part of their contribution to pathology.

The leptomeninges, composed of the arachnoid mater, and pia mater, contains distinct subgroups of fibroblasts that differ in location and transcriptomic profiles. These fibroblasts contribute to the blood–cerebrospinal fluid barrier under physiological conditions, participate in fibrosis, and support blood–brain barrier integrity during injury and disease. However, their Ca²⁺ signaling profiles and underlying mechanisms in health and disease remain poorly understood. In this study, we divided leptomeningeal fibroblasts into three subgroups based on their locations: arachnoid fibroblasts, pia mater fibroblasts and perivascular fibroblasts. We employed two-photon microscopy in awake transgenic mice expressing Ca²⁺ indicators in leptomeningeal fibroblasts to investigate spontaneous and behaviorally evoked Ca²⁺ transients across different fibroblast subgroups. We found that each subgroup exhibits a distinct Ca²⁺ activity profile, with pia mater fibroblasts showing the highest-amplitude Ca²⁺ transients. Moreover, these fibroblasts displayed unique responses to both whisker air-puff stimulation and locomotion. We further demonstrated, using a chronically implanted cannula beneath the cranial window, that locomotion-associated vasodilation is followed by TRPV4 channel-mediated fibroblast Ca²⁺ elevations. Finally, systemic inflammation induced by lipopolysaccharide (LPS) reduced spontaneous Ca²⁺ transients in pia mater fibroblasts, likely due to macrophage infiltration following the inflammatory response. For the first time, this study characterizes spontaneous and behaviorally evoked Ca²⁺ dynamics in distinct leptomeningeal fibroblast subgroups in awake animals, providing novel insights into the functional roles of leptomeningeal fibroblasts in the healthy and diseased brain.