Biology subjects

Vazquez, A. L.

Publications and source records attributed to Vazquez, A. L..

15 recordsLinked to original sources

bioRxiv2026

Astrocyte modulation of neural activity during intracortical microstimulation

Electrical manipulation of neural circuits has advanced our understanding of brain function, but the mechanisms that shape neural activity during stimulation remain incompletely understood. This gap reflects a persistent neuron-centric view of stimulation that overlooks the regulatory influence of glial cells, especially for long duration stimuli. Here we investigated whether astrocytes actively shape the brains response to different patterns of stimulation. Using two-photon and mesoscale calcium imaging of the visual cortex in awake mice, we bidirectionally manipulated astrocyte calcium signaling during intracortical microstimulation (ICMS) via chemogenetic (hM3Dq) and optogenetic (melanopsin) tools. We show that astrocytes respond to stimulation in a pattern-dependent way and shape ongoing neural activity rather than simply following it. Near the electrode, neurons responded to astrocyte modulation in mixed directions, some increasing and some decreasing. Instead of uniformly raising or lowering activity, changing astrocyte calcium pulled neuronal responses toward an intermediate state, with the direction determined by each neurons starting response and its distance from the electrode. Our results identify astrocyte calcium as a source of this shaping during electrical stimulation, and highlight the need to incorporate neuron-glia dynamics into the design of therapeutic neurostimulation.

bioengineering↗

bioRxiv2026

Diverse signaling proteins recruit RCC1-Like Domain proteins to the plasma membrane through a common structural framework.

Plants translate diverse environmental cues into spatially regulated growth responses through intracellular signaling mechanisms that remain incompletely understood. Here, we identify the BREVIS RADIX (BRX) domain of RCC1-LIKE DOMAIN (RLD) proteins as a versatile interaction module that recognizes multiple signaling proteins in Arabidopsis. We identified members of the PHYTOCHROME KINASE SUBSTRATE (PKS) and BIG GRAIN (BG) families as novel RLD interactors families, in addition to the previously characterized LAZY1-LIKE (LZY) proteins and BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL). Structural, biochemical, and cell-based analyses demonstrate that these diverse partners, despite having divergent interaction sequences, bind a common surface on the RLD BRX domain to recruit RLD to the plasma membrane. This interaction relies on a shared {beta}-strand-based recognition mode, in which distinct peptide conformations accommodate binding to the same BRX-domain surface. Furthermore, we show that RLD2 and RLD3 contribute to hypocotyl phototropism, and that disrupting the conserved PKS interaction motif abolishes PKS4 function in this response. Together, our findings identify the RLD BRX domain as a molecular platform for diverse protein interactions and establish a PKS-RLD module that operates in phototropism. Furthermore, these findings strongly suggest that RLD recruitment to the plasma membrane via PKS underlies phototropic signal transduction.

plant biology↗

bioRxiv2026

Linking cross-species trajectories of cerebrovascular remodeling in aging and Alzheimer's disease to brain vessel transcriptome

Cerebrovascular remodeling driven by subtle molecular changes starts early in the asymptomatic stage of Alzheimers disease (AD). Despite progress in human vascular imaging and postmortem tissue analysis, there is limited data on the early features of small vessel reorganization, particularly in the context of cell-specific molecular drivers. This is largely because of the invasive nature of the tools for direct cellular observation and analysis. Since early detection is key, histopathology falls short with end-point data from people that died in late stages of the disease. This is a critical knowledge gap, because the early vascular processes are thought to be strongly correlated with health outcomes, tipping the scales from mild cognitive impairment to AD. To meet these translational challenges, we performed near life-span in vivo two-photon imaging and MRI of the cerebrovascular tree in a mouse model of amyloidosis. We identified precisely when subtle abnormalities in vessel tortuosity and red blood cell velocity first emerge in the context of differential amyloid accumulation in vessels walls and tissues. We then isolated the brain vessels for transcriptional analysis at this flagship timepoint and performed cross-species analysis linking changes in vascular cells to genes and pathways common to both mice and humans. Importantly, using 7T MRI of aging humans, we directly associated vascular remodeling trajectories of mice and humans and identified a remarkably analogous tortuosity course in the smallest brain vessels. Our integrated framework across scales and species advances neuroimaging biomarker understanding and uncovers early mechanistic routs of dysfunctional angiogenesis and actin-mediated contractility.

neuroscience↗

bioRxiv2025

Chronic alteration of Ca2+ and hemodynamic signals induced by intracortical microstimulation in the visual cortex of awake mice

Understanding the chronic effects of intracortical microstimulation (ICMS) and device implantation on cortical function is essential for the development of stable neuroprosthetics. We chronically implanted microelectrode into Thy1-GCaMP6s mice and conducted longitudinal mesoscopic widefield and two-photon Ca2#x002B; imaging alongside intrinsic optical signal recordings over 12 weeks. Six ICMS frequencies (10-500 Hz) and contralateral visual LED stimuli were delivered in repeated sessions. Oxygen extraction fraction (OEF) was estimated from dual-wavelength reflectance, and hemodynamic response functions (HRF) were derived via regularized deconvolution. Over the chronic period, 25-Hz ICMS evoked progressively larger Ca2+ responses whose duration was extending, while spatial spread remained stable around 500 {micro}m. Concurrently, OEF reductions deepened, reflecting increased relative blood supply, and the spatial extent of OEF signals contracted in the first week before expanding by day 21. After discharge-like Ca2+ events emerged in nearly half of mice, predominantly under 25-Hz ICMS. HRF peak amplitude increased between day 0 and days 7-21, with latency decreasing. Chronic ICMS induces progressive potentiation of neuronal and vascular responses alongside local neurovascular uncoupling on day 0 and sustained silencing of spontaneous activity near the implant. These dynamics exhibiting a stabilization 6-7 weeks after the implantation, coupled with frequency-dependent after discharges, underscore the need for adaptive stimulation strategies prior to the stabilization and to prevent the after discharge and implant designs that mitigate local suppression and ensure long-term stability of cortical neuroprostheses.

bioengineering↗

bioRxiv2025

Parvalbumin interneurons mediate spontaneous hemodynamic fluctuations

Resting-state hemodynamic fluctuations are closely linked to gamma-band neural activity, yet the cellular drivers of this neurovascular coupling remain unclear. Given their established contribution in generating gamma oscillations, parvalbumin (PV) interneurons are prime candidates for regulating spontaneous cerebral blood flow (CBF) dynamics. Using chemogenetic tools in awake PV-Cre mice, we modulated PV interneuron activity and measured effects on neural network activity, local field potentials (LFP), and hemodynamics. Two-photon (2P) calcium imaging confirmed effective PV modulation, which affected excitatory neuron activity. PV suppression reduced high-gamma power, increased low-frequency LFP activity, and elevated basal CBF. 2P vessel imaging showed increased basal arterial diameter and significantly greater diameter fluctuation power in deeper cortical layers enriched with PV cells, but not in superficial layers. PV suppression also significantly weakened the correlation between gamma LFP power and CBF. Despite increased apparent neuronal synchrony during PV suppression, its relationship to arterial dynamics remained stable, possibly due to compensatory regulation by subsets of PV-positive and PV-negative cells. These findings provide causal evidence that PV interneuron contribute to spontaneous neurovascular dynamics and mediate the link between gamma oscillations and resting-state hemodynamic signals, revealing their significant role in maintaining functional connectivity and vascular regulation during non-task-engaged brain states.

neuroscience↗

bioRxiv2025

Intracortical Microstimulation Induces Rapid Microglia Process Convergence

Intracortical microstimulation (ICMS) has demonstrated the potential to restore vision and hearing by stimulating relevant cortical regions in both animals and humans, offering significant clinical promise for sensory restoration. While the neuronal response to ICMS has been extensively studied at the cellular level through electrophysiology and two-photon (2P) imaging, the response of non-neuronal cells, particularly microglia, as well as the effects of ICMS on blood-brain barrier (BBB) integrity remain poorly understood. In this study, we applied ICMS under 2P imaging in dual-reporter mice, with green fluorescent protein labeling microglia and a red fluorescent Ca2+ indicator labeling neurons. We also monitored vascular dye leakage to assess BBB integrity throughout the experiment. Using clinically relevant waveform parameters, we tested a range of current amplitudes. Surprisingly, we found that microglia responded rapidly, within 15 minutes of stimulation, by converging their processes (MPC) on areas of high neural activity. The prevalence of MPC increased with higher current amplitudes, but intriguingly, it did not correlate with the strength of the local electric field. Additionally, vascular dye penetration into brain tissue was higher in stimulated animals than in controls and increased with current amplitude. This study reveals a rapid microglia and BBB response to ICMS that has not been reported previously, underscoring the need for further research to fully characterize the biological response to ICMS and establish improved safety standards.

bioengineering↗

bioRxiv2024

Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing

Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy (FCS), we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least two-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/619117v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@11a1f94org.highwire.dtl.DTLVardef@f3130org.highwire.dtl.DTLVardef@1666938org.highwire.dtl.DTLVardef@85fd13_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Spatiotemporal properties of cortical excitatory and inhibitory neuron activation by sustained and bursting electrical microstimulation

Intracortical microstimulation (ICMS) of sensory cortices produces artificial sensation yet the neural mechanisms underlying evoked responses, particularly among inhibitory subpopulations, remain unclear. We investigated how long durations (30 s) of ICMS shape spatiotemporal patterns in excitatory and inhibitory network activation using two-photon imaging of visual cortex in transgenic mice. Inhibitory neurons were more likely to facilitate (increase in activation) across 30 s of ICMS, whereas excitatory neurons were more likely to adapt (decrease in activation) and exhibit post-ICMS depression. Different temporal profiles led to preferential activation of excitatory or inhibitory neurons, with theta-burst stimulation driving the strongest inhibitory response and 10-Hz burst patterns driving the strongest peak excitatory response. Neurons located farther from the electrode exhibited more diverse responses to ICMS, highlighting synaptic recruitment dynamics such as inhibition and disinhibition. This study reveals how ICMS differentially influences excitatory and inhibitory neuron activity across long durations of ICMS and suggests temporal patterning can be used to potentially target neuronal subpopulations and drive desirable activity patterns.

bioengineering↗

bioRxiv2024

Parvalbumin interneuron activity induces slow cerebrovascular fluctuations in awake mice

Neuronal regulation of cerebrovasculature underlies brain imaging techniques reliant on cerebral blood flow (CBF) changes. However, interpreting these signals requires understanding their neural correlates. Parvalbumin (PV) interneurons are crucial in network activity, but their impact on CBF is not fully understood. Optogenetic studies show that stimulating cortical PV interneurons induces diverse CBF responses, including rapid increases, decreases, and slower delayed increases. To clarify this relationship, we measured hemodynamic and neural responses to optogenetic stimulation of PV interneurons expressing Channelrhodopsin-2 during evoked and ongoing resting-state activity in the somatosensory cortex of awake mice. Two-photon microscopy (2P) Ca2+ imaging showed robust activation of PV-positive (PV+) cells and inhibition of PV-negative (PV-) cells. Prolonged PV+ cell stimulation led to a delayed, slow CBF increase, resembling a secondary peak in the CBF response to whisker stimulation. 2P vessel diameter measurements revealed that PV+ cell stimulation induced rapid arterial vasodilation in superficial layers and delayed vasodilation in deeper layers. Ongoing activity recordings indicated that both PV+ and PV-cell populations modulate arterial fluctuations at rest, with PV+ cells having a greater impact. These findings show that PV interneurons generate a complex depth-dependent vascular response, dominated by slow vascular changes in deeper layers.

neuroscience↗

bioRxiv2024

Potential of Photoelectric Stimulation with Ultrasmall Carbon Electrode on Neural Tissue: New Directions in Neuromodulation Technology Development

ObjectiveNeuromodulation technologies have gained considerable attention for its clinical potential in treating neurological disorders and their capacity to advance cognition research. Nevertheless, traditional neuromodulation methods such as electrical stimulation and optogenetics manipulation currently experience technical and biological challenges that hinge their therapeutic potential and chronic research applications. Recently, a promising alternative neuromodulation approach based on the photoelectric effect has emerged. This approach is capable of generating electrical pulses when exposed to near-infrared (NIR) light and allows modulation of neuronal activity without the need for genetic alterations. In this study, we investigate a variety of design strategies aimed at enhancing photoelectric stimulation using minimally invasive, ultrasmall, untethered carbon electrodes. ApproachA multiphoton laser was employed as the NIR light source. Benchtop investigations were conducted using a three-electrode setup, and chronopotentiometry was used to record photo-stimulated voltage. For in vivo evaluation, we used Thy1-GCaMP6s mice with acute implantation of ultrasmall carbon electrodes. Main resultsWe revealed the beneficial effects of high duty-cycle laser scanning and photovoltaic polymer interfaces on the photo-stimulated voltages of ultrasmall carbon electrodes. Additionally, we demonstrated the promising potential of carbon-based diamond electrodes for photoelectric stimulation and examined the application of photoelectric stimulation in precise chemical delivery by loading mesoporous silica nanoparticles (SNPs) co-deposited with polyethylenedioxythiophene (PEDOT). SignificanceThese findings on photoelectric stimulation utilizing ultrasmall carbon electrodes underscore its immense potential for advancing the next generation of neuromodulation technology. This approach offers the opportunity to effectively modulate neural tissue while minimizing invasive implantation-related injuries in freely moving subjects, which hold significant promise for a wide range of applications in neuroscience research and clinical settings.

bioengineering↗

bioRxiv2024

Activation and depression of neural and hemodynamic responses induced by the intracortical microstimulation and visual stimulation in the mouse visual cortex.

Objective. Intracortical microstimulation can be an effective method for restoring sensory perception in contemporary brain-machine interfaces. However, the mechanisms underlying better control of neuronal responses remain poorly understood, as well as the relationship between neuronal activity and other concomitant phenomena occurring around the stimulation site. Approach. Different microstimulation frequencies were investigated in vivo on Thy1-GCaMP6s mice using widefield and two-photon imaging to evaluate the evoked excitatory neural responses across multiple spatial scales as well as the induced hemodynamic responses. Specifically, we quantified stimulation-induced neuronal activation and depression in the mouse visual cortex and measured hemodynamic oxyhemoglobin and deoxyhemoglobin signals using mesoscopic-scale widefield imaging. Main results. Our calcium imaging findings revealed a preference for lower-frequency stimulation in driving stronger neuronal activation. A depressive response following the neural activation preferred a slightly higher frequency stimulation compared to the activation. Hemodynamic signals exhibited a comparable spatial spread to neural calcium signals. Oxyhemoglobin concentration around the stimulation site remained elevated during the post-activation (depression) period. Somatic and neuropil calcium responses measured by two-photon microscopy showed similar dependence on stimulation parameters, although the magnitudes measured in soma was greater than in neuropil. Furthermore, higher-frequency stimulation induced a more pronounced activation in soma compared to neuropil, while depression was predominantly induced in soma irrespective of stimulation frequencies. Significance. These results suggest that the mechanism underlying depression differs from activation, requiring ample oxygen supply, and affecting neurons. Our findings provide a novel understanding of evoked excitatory neuronal activity induced by intracortical microstimulation and offer insights into neuro-devices that utilize both activation and depression phenomena to achieve desired neural responses.

bioengineering↗

bioRxiv2022

Long-range inhibitory neurons mediate cortical neurovascular coupling

Neuronal activity evokes a vascular response that is essential to sustain brain function. We show that neurovascular coupling (NVC) is mediated by long-range projecting GABAergic neurons that express Tacr1. Whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feed-forward excitatory pathways. Optogenetic activation of Tacr1 neurons elicited vasodilation, whereas inhibition significantly reduced whisker-evoked hemodynamic responses. Moreover, vasodilation was preceded by capillary pericyte activity, demonstrating a mechanism for NVC.

neuroscience↗

bioRxiv2022

Integrated microprism and micro-electrode array for chronic in vivo two-photon imaging and electrophysiology across all cortical layers

Electrophysiology is a vital tool in neuroscience research with increasing translational value. It is used to record or modulate neuronal activity with high temporal but lower spatial resolution. Optical technologies, such as two-photon microscopy (TPM) can complement electrophysiological recordings with large-scale imaging at cellular resolution. Combining these two provides a powerful platform to elucidate and coordinate multimodal functions. Prior attempts have been limited to the superficial brain from a top-down optical view. Here, we describe a novel combination of transparent microelectrode arrays (MEAs) with glass microprisms for simultaneous electrophysiology and optical imaging of all cortical layers in a vertical plane. We tested our device in Thy1-GCaMP6 mice for over 4 months and demonstrated its capability for multisite single-unit recording, microstimulation, and simultaneous TPM calcium imaging. Using this setup, we reveal how amplitude, frequency, and depth of microstimulation impact neural activation patterns across the cortical column. This work presents a multimodal tool that extends integrated electrophysiology and optical imaging from the superficial brain to the whole cortical column, opening new avenues of neuroscience research and neurotechnology development.

bioengineering↗

bioRxiv2018

Calcium activation of frequency dependent temporally phasic, localized, and dense population of cortical neurons by continuous electrical stimulation

Electrical stimulation of the brain has become a mainstay of fundamental neuroscience research and an increasingly prevalent clinical therapy. Despite decades of use in basic neuroscience research and the growing prevalence of neuromodulation therapies, gaps in knowledge regarding activation or inactivation of neural elements over time have limited its ability to adequately interpret evoked downstream responses or fine-tune stimulation parameters to focus on desired responses. In this work, in vivo two-photon microscopy was used to image neuronal calcium activity in layer 2/3 neurons of somatosensory cortex (S1) in male C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J mice during 30 s of continuous electrical stimulation at varying frequencies. We show frequency-dependent differences in spatial and temporal somatic responses during continuous stimulation. Our results elucidate conflicting results from prior studies reporting either dense spherical activation of somas biased towards those near the electrode, or sparse activation of somas at a distance via axons near the electrode. These findings indicate that the neural element specific temporal response local to the stimulating electrode changes as a function of applied charge density and frequency. These temporal responses need to be considered to properly interpret downstream circuit responses or determining mechanisms of action in basic science experiments or clinical therapeutic applications.\n\nSignificance StatementMicrostimulation of small populations of neurons has the potential to ameliorate symptoms associated with several neurological disorders. However, the specific mechanisms by which microstimulation elicits therapeutic responses are unclear. This work examines the effects of continuous microstimulation on the local population of neurons surrounding the implanted electrode. Stimulation was found to elicit spatiotemporal neuronal responses in a frequency dependent manner. These findings suggest that stimulation frequency may be an important consideration for applications in research or therapy. Further research aimed at understanding these neuronal activation properties may provide insight into the mechanistic mode of action of continuous microstimulation.

neuroscience↗

bioRxiv2018

Viral-mediated optical stimulation of peripheral motor nerves in non-human primates

ObjectiveReanimation of muscles paralyzed by disease states such as spinal cord injury remains a much sought after therapeutic goal of neuroprosthetic research. Optogenetic stimulation of peripheral motor nerves expressing light-sensitive opsins is a promising approach to muscle reanimation that may overcome several drawbacks of traditional methods such as functional electrical stimulation (FES). However, the utility of these methods has only been demonstrated in rodents to date, while translation to clinical practice will likely first require demonstration and refinement of these gene therapy techniques in non-human primates.\n\nApproachThree rhesus macaques were injected intramuscularly with either one or both of two optogenetic constructs (AAV6-hSyn-ChR2-eYFP and/or AAV6-hSyn-Chronos-eYFP) to transduce opsin expression in the corresponding nerves. Neuromuscular junctions were targeted for virus delivery using an electrical stimulating injection technique. Functional opsin expression was periodically evaluated up to 13 weeks post-injection by optically stimulating targeted nerves with a 472 nm fiber-coupled laser while recording electromyographic (EMG) responses.\n\nMain ResultsOne monkey demonstrated functional expression of ChR2 at 8 weeks post-injection in each of two injected muscles, while the second monkey briefly exhibited contractions coupled to optical stimulation in a muscle injected with the Chronos construct at 10 weeks. A third monkey injected only in one muscle with the ChR2 construct showed strong optically coupled contractions at 5 [1/2] weeks which then disappeared by 9 weeks. EMG responses to optical stimulation of ChR2-transduced nerves demonstrated graded recruitment relative to both stimulus pulse-width and light intensity, and were able to track stimulus trains up to 16 Hz. In addition, the EMG response to prolonged stimulation showed delayed fatigue over several minutes.\n\nSignificanceThese results demonstrate the feasibility of viral transduction of peripheral motor nerves for functional optical stimulation of motor activity in non-human primates, a variable timeline of opsin expression in a primate model closer to humans, and fundamental EMG response characteristics to optical nerve stimulation. Subsequently, they represent an important step in translating these optogenetic techniques as a clinically viable gene therapy.

bioengineering↗