Biology subjects

Kleinfeld, D.

Publications and source records attributed to Kleinfeld, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Neurons entrain and shape an intrinsic arteriole rhythm that distributes blood across the cortical mantle

The cerebral arteriole network distributes blood across cortex through branches whose diameters oscillate near a frequency fvaso ~ 0.1 Hz. We asked whether the spatiotemporal pattern of these vaso-oscillations reflects neuronal entrainment of an intrinsic vascular oscillation. Three findings support this: First, suppressing endothelial hyperpolarization left resting-state oscillations intact but weakened the vascular response to neural activity, showing that vasomotion originates within the arteriole network rather than following neuronal drive. Second, arterioles amplify sensory drive selectively rather than faithfully: neural activity near 0.1 Hz is resonantly enhanced to form a strong peak in the vascular response. Third, neuronal frequency sets spatial reach, and areas compete: when two neighboring cortical areas are driven simultaneously, the one nearer fvaso captures more vascular territory. Neurons therefore entrain an intrinsic vascular rhythm and stimulation frequency governs how vasomotion distributes blood, shapes functional brain images, and may influence interstitial fluid transport.

neuroscience↗

bioRxiv2026

Calcium-independent biosensors for multiplexed in vivo imaging of extracellular lactate dynamics

Extracellular L-lactate reports cellular metabolism, but resolving its dynamics alongside intracellular metabolism and neuronal activity remains challenging. Here we develop eLACCO3 and R-eLACCO3, green and red cell-surface lactate biosensors with Ca2+-independent responses and faster response and recovery kinetics in cultured cells. Selected variants provide larger fluorescence responses than their predecessors in brain cells, slices, and living mice and improve detection of glucose-evoked extracellular lactate oscillations. Combining R-eLACCO3 with GCaMP6f enabled imaging of extracellular L-lactate simultaneously with odor-evoked activity in anesthetized mice and cortical activity in awake mice. These biosensors enable multiplexed measurements of extracellular lactate dynamics in relation to intracellular metabolism and neuronal activity.

molecular biology↗

bioRxiv2026

Axons organize into micro-tracts around the cortical vasculature

Neural activity is metabolically costly and supported by local increases in cerebral blood flow. The process by which neural activity drives blood flow increase, neurovascular coupling (NVC), remains poorly understood. One persistent knowledge gap is how subcellular compartments are arranged at the vascular wall, where local signaling occurs. This requires a survey of the neurovascular interface at the nanometer scale. To this end, we analyzed a ~1 mm3 volume of mouse visual cortex imaged with serial electron microscopy. We manually labeled different vascular zones, and classified the EM segments surrounding hundreds of vessel segments across these zones. We found that capillaries and venous vessels are surrounded by more axonal volume than arteriolar vessels. Perivascular axons tended to bundle into micro-tracts, where axons ran in parallel and coursed near the vessel wall in a variety of geometric orientations. Micro-tracts cover ~60% of the surface of capillaries, but only 24-40% of every other vascular zone's surface. Finally, we show that the extensive axon arbors of basket cell inhibitor neurons are within micro-tracts. Their axons come in closest proximity to vessels, and on average, approach 2 to 3 fold more vessel segments than other neuronal subtypes. Altogether, perivascular axonal micro-tracts may be an important physical substrate for NVC signaling at capillaries.

neuroscience↗

bioRxiv2026

Ultralow frequency vaso-oscillations in human cerebral arteries are independent from Mayer waves

This study tests the hypothesis that vasomotion, an [~] 0.1 Hz oscillation in arteriole diameter, is generated by intrinsic oscillations within the arterioles that perfuse the brain, and not by external drive from systemic blood pressure oscillations (Mayer waves). During cardio-pulmonary bypass that transiently eliminated systemic blood pressure oscillations in 14 patients, we observed that vasomotor oscillations persist with normal amplitudes and frequencies over the one- to three-hour time course of surgery. In contrast, [~] 0.1 Hz oscillations in peripheral blood pressure were predominantly absent. This implies that cerebral arterioles generate their own rhythmic vaso-dynamics, although we cannot discount that vasomotion can phase-lock with [~] 0.1 Hz systemic physiological rhythms in the awake, healthy state. We discuss the impact of this finding on the role of vasomotion in modulating the perfusion of blood and the transport of interstitial fluid in the brain.

neuroscience↗

bioRxiv2026

A hypoxia-sensitive medullary nucleus modulates cerebral blood flow via a disynaptic pathway to cortex

The RVLM (rostral ventral lateral medulla) region of the brainstem is implicated as a controller of both systemic and cerebral blood flow (CBF). Past studies leave open the question of how the RVLM stabilizes CBF in awake animals. Here, we focus on CBF regulation by the adenergic subpopulation of RVLM neurons (RVLMD{beta}h). Hypoxic challenge increases the variability of the activity of RVLMD{beta}h neurons along with increased CBF. Experimental photoactivation of RVLMD{beta}h neurons leads to rapid vasodilation of pial arterioles across the cortical mantle and increased CBF, which is only then followed by an increase in cortical activity. No significant changes in systemic physiology are observed. Virus tracing establishes disynaptic pathways from the RVLM to neocortex with predominant relays involving the lateral hypothalamus and the zona incerta subthalamic nuclei. Chemogenetic inhibition of those nuclei led to a 70 % reduction in the ability of photoactivated RVLMD{beta}h neurons to induce cortical vasodilation and increase CBF. In toto, these findings reveal a major RVLM subcortical pathway to drive transcortical increases in CBF and represent an adaptive mechanism to inform the cerebral vasculature about environmental shifts in pO2.

neuroscience↗

bioRxiv2025

Spectral disruption of inter-hemispheric resting-state BOLD coherence at vasomotor frequency (~0.1 Hz) links vascular dysfunction to functional connectivity loss in carotid artery stenosis

In the brain, vasomotor dynamics at infra-slow frequencies ([~]0.1 Hz), driven by synchronized oscillations of smooth muscle cells in vessel walls, are thought to play a crucial role in regulating cerebral perfusion and underlie resting-state functional connectivity (FC), typically measured by correlated time courses of functional signals. In particular, rodent studies have demonstrated that vasomotor activity contributes to the coherence of blood oxygenation level dependent (BOLD) signal fluctuations. However, in humans, detecting this contribution non-invasively remains challenging due to the limited spatiotemporal sensitivity of functional magnetic resonance imaging (fMRI) to vasomotion. Given that prior studies have identified internal carotid artery stenosis (ICAS) as an informative conditional lesion model of vasomotor and hemodynamic impairments in humans, we investigated whether ICAS affects interhemispheric BOLD coherence at [~]0.1 Hz. Using a multi-modal fMRI framework integrating resting-state fMRI with quantitative mapping of cerebral blood volume, blood flow, oxygen metabolism, and BOLD time lag, we compared BOLD coherence between patients with asymptomatic unilateral ICAS and healthy controls. Frequency-specific analysis revealed significantly diminished interhemispheric BOLD coherence at [~]0.1 Hz across canonical resting-state networks in ICAS patients, while ultra-slow (<0.05 Hz) coherence remained largely preserved. This reduction was spatially widespread and particularly pronounced in watershed areas, i.e., border zones between major vascular territories, associated with significantly increased lateralization of cerebral blood volume (p < 0.01). Notably, coherence-based FC patterns at [~]0.1 Hz were heterogeneous within watershed areas but homogeneous outside, suggesting an interplay between compensatory mechanisms and cerebrovascular impairment. Taken together, our findings demonstrate that ICAS induces subtle, frequency-and region-specific alterations in interhemispheric FC, consistent with a model in which impaired vasomotor activity impacts on resting-state FC in the human brain.

neuroscience↗

bioRxiv2025

Glutamate indicators with increased sensitivity and tailored deactivation rates

Identifying the input-output operations of neurons requires measurements of synaptic transmission simultaneously at many of a neurons thousands of inputs in the intact brain. To facilitate this goal, we engineered and screened 3365 variants of the fluorescent protein glutamate indicator iGluSnFR3 in neuron culture, and selected variants in the mouse visual cortex. Two variants have high sensitivity, fast activation (< 2 ms) and deactivation times tailored for recording large populations of synapses (iGluSnFR4s, 153 ms) or rapid dynamics (iGluSnFR4f, 26 ms). By imaging action-potential evoked signals on axons and visually-evoked signals on dendritic spines, we show that iGluSnFR4s/4f primarily detect local synaptic glutamate with single-vesicle sensitivity. The indicators detect a wide range of naturalistic synaptic transmission, including in the vibrissal cortex layer 4 and in hippocampal CA1 dendrites. iGluSnFR4 increases the sensitivity and scale (4s) or speed (4f) of tracking information flow in neural networks in vivo.

neuroscience↗

bioRxiv2025

A brainstem map of orofacial rhythms

Rhythmic orofacial movements, such as eating, drinking, or vocalization, are controlled by distinct premotor oscillator networks in the brainstem. Orofacial movements must be coordinated with rhythmic breathing to avoid aspiration and because they share muscles. Understanding how brainstem circuits coordinate rhythmic motor programs requires neurophysiological measurements in behaving animals. We used Neuropixels probe recordings to map brainstem neural activity related to breathing, licking, and swallowing in mice drinking water. Breathing and licking rhythms were tightly coordinated and phase-locked, whereas intermittent swallowing paused breathing and licking. Multiple clusters of neurons, each recruited during different orofacial rhythms, delineated a lingual premotor network in the intermediate nucleus of the reticular formation (IRN). Local optogenetic perturbation experiments identified a region in the IRN where constant stimulation can drive sustained rhythmic licking, consistent with a central pattern generator for licking. Stimulation to artificially induce licking showed that coupled brainstem oscillators autonomously coordinated licking and breathing. The brainstem oscillators were further patterned by descending inputs at moments of licking initiation. Our results reveal the logic governing interactions of orofacial rhythms during behavior and outline their neural circuit dynamics, providing a model for dissecting multi-oscillator systems controlling rhythmic motor programs.

neuroscience↗

bioRxiv2024

Neurovascular Impulse Response Function (IRF) during spontaneous activity differentially reflects intrinsic neuromodulation across cortical regions

Ascending neuromodulatory projections from deep brain nuclei generate internal brain states that differentially engage specific neuronal cell types. Because neurovascular coupling is cell-type specific and neuromodulatory transmitters have vasoactive properties, we hypothesized that the impulse response function (IRF) linking spontaneous neuronal activity with hemodynamics would depend on neuromodulation. To test this hypothesis, we used optical imaging to measure (1) release of neuromodulatory transmitters norepinephrine (NE) or acetylcholine (ACh), (2) Ca2+ activity of local cortical neurons, and (3) changes in hemoglobin concentration and oxygenation across the dorsal surface of cerebral cortex during spontaneous neuronal activity in awake mice. A canonical convolution model with a global, stationary IRF (i.e., the convolution kernel) describing evolution of total hemoglobin (HbT, reflective of dilation dynamics) with respect to Ca2+, resulted in a poor fit to the data. However, the HbT time-course was well predicted, pixel-by-pixel, by a weighted sum of Ca2+ and NE time-courses. Consistent with this result, modeling HbT as a weighted sum of stationary Ca2+ - and NE-specific IRFs (IRFCa2+ and IRFNE) convolved with the respective time-courses dramatically improved the fit compared to the global IRF. IRFCa2+ and IRFNE, estimated from the data, were positive and negative, respectively. In contrast to NE, ACh was largely redundant with Ca2+ and therefore did not improve HbT 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 while neurovascular coupling with respect to neuronal Ca2+ is a dynamic and seemingly complex phenomenon, hemodynamic fluctuations can be captured by a simple linear model with stationary IRFs with respect to the underlying dilatory and constrictive forces. In the current study, these forces were captured by the positive IRFCa2+ (dilation) and negative IRFNE (constriction). Without accounting for NE neuromodulation and the associated vasoconstriction, diminished hemodynamic coherence, commonly referred to as "functional (dys)connectivity" in BOLD fMRI studies, can be falsely interpreted as neuronal desynchronizations.

neuroscience↗

bioRxiv2023

Machine assisted annotation in neuroanatomy

One of the important yet labor intensive tasks in neuroanatomy is the identification of select populations of cells. Current high-throughput techniques enable marking cells with histochemical fluorescent molecules as well as through the genetic expression of fluorescent proteins. Modern scanning microscopes allow high resolution multi-channel imaging of the mechanically or optically sectioned brain with thousands of marked cells per square millimeter. Manual identification of all marked cells is prohibitively time consuming. At the same time, simple segmentation algorithms suffer from high error rates and sensitivity to variation in fluorescent intensity and spatial distribution. We present a methodology that combines human judgement and machine learning that serves to significantly reduce the labor of the anatomist while improving the consistency of the annotation. As a demonstration, we analyzed murine brains with marked premotor neurons in the brainstem. We compared the error rate of our method to the disagreement rate among human anatomists. This comparison shows that our method can reduce the time to annotate by as much as ten-fold without significantly increasing the rate of errors. We show that our method achieves significant reduction in labor while achieving an accuracy that is similar to the level of agreement between different anatomists.

neuroscience↗

bioRxiv2023

Spatiotemporal development of growth and death zones in expanding bacterial colonies driven by emergent nutrient dynamics

Bacterial colony growth on hard agar is commonplace in microbiology; yet, what occurs inside a growing colony is complex even in the simplest cases. Robust colony expansion kinetics featuring a linear radial growth and a saturating vertical growth indicates a common developmental program which is elucidated here for Escherichia coli cells using a combination of modeling and experiments. Radial colony expansion is found to be limited by mechanical factors rather than nutrients as commonly assumed. In contrast, vertical expansion is limited by glucose depletion inside the colony, an effect compounded by reduced growth yield due to anaerobiosis. Carbon starvation in the colony interior results in substantial cell death within 1-2 days, with a distinct death zone that expands with the growing colony. Overall, the development of simple colonies lacking EPS production and differentiation is dictated by an interplay of mechanical constraints and emergent nutrient gradients arising from obligatory metabolic processes.

microbiology↗

bioRxiv2022

A change in behavioral state switches the pattern of motor output that underlies rhythmic head and orofacial movements

The breathing rhythm serves as a reference that paces orofacial motor actions and orchestrates active sensing. Past work reports that pacing occurs solely at a fixed phase relative to sniffing. We reevaluated this constraint as a function of exploratory behavior. Allocentric and egocentric rotations of the head and the electromyogenic activity of the underlying motoneurons for head and orofacial movements were recorded in free-ranging rats as they searched for food. We found that a change in state from foraging to rearing is accompanied by a change in the phase of muscular activation relative to sniffing, so that pacing now occurs at one of two phases. Further, head-turning is biased such that an animal gathers a novel sample of its environment upon inhalation. In toto, the coordination of active sensing has a previously unrealized computational complexity that, in principle, can emerge from hindbrain circuits with fixed architecture and credible synaptic time-delays.

neuroscience↗

bioRxiv2022

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

neuroscience↗

bioRxiv2021

Cortex-independent open-loop control of a voluntary orofacial motor action

Whether using our eyes or our hands, we interact with our environment through mobile sensors. The efficient use of these sensory organs implies the ability to track their position; otherwise, perceptual stability and prehension would be profoundly impeded1,2. The nervous system may be informed about the position of a sensory organ via two complementary feedback mechanisms: peripheral reafference (external, sensory feedback) and efference copy (internal feedback)3-6. Yet, the potential contributions of these mechanisms remain largely unexplored. By training rats to place their vibrissae within a predetermined angular range without contact, a task that depends on knowledge of vibrissa position relative to their face, we found that peripheral reafference is not required. The presence of motor cortex is not required either, even in the absence of peripheral reafference. On the other hand, the red nucleus, which receives descending inputs from motor cortex and the cerebellum and projects to facial motoneurons7-10, is critical for the execution of the vibrissa task. All told, our results demonstrate the existence of an open-loop control by an internal model that is sufficient to drive voluntary motion. The internal model is independent of motor cortex and likely contains the cerebellum and associated nuclei.

neuroscience↗

bioRxiv2021

Probing neuropeptide volume transmission in vivo by a novel all-optical approach

Neuropeptides are essential signaling molecules in the nervous system involved in modulating neural circuits and behavior. Although hypothesized to signal via volume transmission through G-protein coupled receptors (GPCR), remarkably little is known about their extrasynaptic diffusion. Here, we developed an all-optical approach to probe neuropeptide volume transmission in mouse neocortex. To control neuropeptide release, we engineered photosensitive nanovesicles with somatostatin-14 (SST) that is released with near-infrared light stimulation. To detect SST, we created a new cell-based neurotransmitter fluorescent engineered reporter (CNiFER) using the SST2 GPCR. Under two-photon imaging, we determined the time to activate SST2R at defined distances as well as the maximal distance and loss rate for SST volume transmission in neocortex. Importantly, we determined that SST transmission is significantly faster in neocortex with a chemically degraded extracellular matrix, a diseased condition indicated in neuroinflammation and Parkinsons disease. These new neurotechnologies can reveal important biological signaling processes previously not possible, and provide new opportunities to investigate volume transmission in the brain.

neuroscience↗

bioRxiv2021

A vibrissa pathway that activates the limbic system

Vibrissa sensory inputs play a central role in driving rodent behavior. These inputs transit through the sensory trigeminal nuclei, which give rise to the ascending lemniscal and paralemniscal pathways. While lemniscal projections are somatotopically mapped from brain stem to cortex, those of the paralemniscal pathway are more widely distributed. Yet the extent and topography of paralemniscal projections are unknown, along with the potential role of these projections in controlling behavior. Here we used viral tracers to map paralemniscal projections. We find that this pathway broadcasts vibrissa-based sensory signals to brain stem regions that are involved in the regulation of autonomic functions and to forebrain regions that are involved in the expression of emotional reactions. We further provide evidence that GABAergic cells of the Kolliker-Fuse nucleus gate trigeminal sensory input in the paralemniscal pathway via a mechanism of presynaptic or extrasynaptic inhibition.

neuroscience↗

bioRxiv2021

Constructing An Adult Orofacial Premotor Atlas In Allen Mouse CCF

Premotor circuits in the brainstem control pools of orofacial motoneurons to execute essential functions such as drinking, eating, breathing, and in rodent, whisking. Previous transsynaptic tracing studies only mapped orofacial premotor circuits in neonatal mice but the adult circuits remain unknown due to technical difficulties. Here we developed a three-step monosynaptic transsynaptic tracing strategy to identify premotor neurons controlling whisker, tongue protrusion, and jaw-closing muscles in the adult. We registered these different groups of premotor neurons onto the Allen mouse brain common coordinate framework (CCF) and consequently generated a combined 3D orofacial premotor atlas, revealing unique spatial organizations of distinct premotor circuits. We also uncovered premotor neurons simultaneously innervating multiple motor nuclei and, thus, likely coordinating different muscles involved in the same orofacial behaviors. Our method for tracing adult premotor circuits and registering to Allen CCF is generally applicable and should facilitate the investigations of motor controls of diverse behaviors.

neuroscience↗

bioRxiv2020

Modulating the Blood-Brain Barrier by Light Stimulation of Molecular-Targeted Nanoparticles

The blood-brain barrier (BBB) tightly regulates the entry of molecules into the brain by tight junctions that seals the paracellular space and receptor-mediated transcytosis. It remains elusive to selectively modulate these mechanisms and to overcome BBB without significant neurotoxicity. Here we report that light stimulation of tight junction-targeted plasmonic nanoparticles selectively opens up the paracellular route to allow diffusion through the compromised tight junction and into the brain parenchyma. The BBB modulation does not impair vascular dynamics and associated neurovascular coupling, or cause significant neural injury. It further allows antibody and adeno-associated virus delivery into local brain regions. This novel method offers the first evidence of selectively modulating BBB tight junctions and opens new avenues for therapeutic interventions in the central nervous system. One Sentence SummaryGentle stimulation of molecular-targeted nanoparticles selectively opens up the paracellular pathway and allows macromolecules and gene therapy vectors into the brain.

neuroscience↗