Cerebellar Purkinje cells can differentially modulate coherence between sensory and motor cortex depending on region and behavior

Activity of sensory and motor cortices is essential for sensorimotor integration. In particular, coherence between these areas may indicate binding of critical functions like perception, motor planning, action, or sleep. Evidence is accumulating that cerebellar output modulates cortical activity and coherence, but how, when, and where it does so is unclear. We studied activity in and coherence between S1 and M1 cortices during whisker stimulation in the absence and presence of optogenetic Purkinje cell stimulation in crus 1 and 2 of awake mice, eliciting strong simple spike rate modulation. Without Purkinje cell stimulation, whisker stimulation triggers fast responses in S1 and M1 involving transient coherence in a broad spectrum. Simultaneous stimulation of Purkinje cells and whiskers affects amplitude and kinetics of sensory responses in S1 and M1 and alters the estimated S1–M1 coherence in theta and gamma bands, allowing bidirectional control dependent on behavioral context. These effects are absent when Purkinje cell activation is delayed by 20 ms. Focal stimulation of Purkinje cells revealed site specificity, with cells in medial crus 2 showing the most prominent and selective impact on estimated coherence, i.e., a strong suppression in the gamma but not the theta band. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1–M1 phase consistency predominantly via ventrolateral thalamus and M1. Our results indicate that activity of sensorimotor cortices can be dynamically and functionally modulated by specific cerebellar inputs, highlighting a widespread role of the cerebellum in coordinating sensorimotor behavior.

Integration of sensory feedback into motor control is of obvious importance for the learning and execution of skilled movements. This integration is particularly relevant when subjects explore their environment via active touch, requiring sensory input to be directly related to the momentary position and movement of eyes, fingertips, antennae, whiskers, or other sense organs (1–3). In vertebrates, adapting movement based upon somatosensory feedback requires collaborative action of primary somatosensory (S1) and motor (M1) cortices (4–6), which are directly and reciprocally connected (7–12). The reciprocal connections between S1 and M1 imply that each region can differentially affect the amplitude of neuronal responses in the other region. These connections can also create coherent activity patterns in S1 and M1 (13–17).Coherence can add an extra layer of neuronal integration, as it has been suggested to bind different brain areas by affecting susceptibility of neurons to synaptic input and providing a timing mechanism for generating a common dynamical frame for cortical operations (18–22). As such, coherence can create a temporal framework for concerted neural activity that facilitates integration of the activity of sensory and motor areas (23–25). Coherence often occurs in specific frequency bands that can be associated with different functions. In the field of sensorimotor integration, skilled movements rely on intercortical coherence between sensory and motor areas that occur in the theta range (4 to 8 Hz) during force generation, while coherence at higher bands is engaged during the preparation thereof (26). Likewise, within the field of visual perception, coherence in the alpha (8 to 12 Hz) and gamma (30 to 100 Hz) bands have been found to contribute to feedback and feedforward processing, respectively (27, 28).Cortical coherence can be modulated by subcortical activity, as has been particularly well established for the thalamus (29–32). Accordingly, the cerebellum, one of the main inputs to the thalamus, has a strong impact in organizing cortical coherence (33–35). Indeed, disruption of cerebellar function, whether inflicted pharmacologically in rats (34) or by stroke in patients (33), affects cortico-cortical coherence.Given the strong cerebello-thalamo-cortical projections (36, 37), it is not surprising that cerebellar activity affects the amplitude and coherence of S1 and M1 (20, 34, 38, 39). However, it is unclear to what extent cerebellar activity can modulate the kinetics of the fast sensory responses in S1 that are propagated by the canonical lemniscal and paralemniscal pathways (40), whether it can differentially influence individual frequency bands, to what extent such potentially different impacts depend on the behavioral context, and whether these differential effects are mediated through different cerebellar modules (41–43). Here, we set out to address these questions by investigating the impact of Purkinje cell activity on the response amplitude and the estimated coherence between the whisker areas of the primary somatosensory (wS1) and motor cortex (wM1) during stimulation of the whiskers in awake behaving mice. Stimulating the whiskers results in transient sensory responses in wS1 and wM1, which were affected by optogenetic stimulation of Purkinje cells at different intervals with respect to air-puff stimulation of the whiskers. The estimated coherence of the sensory response in wS1 and wM1 was differentially modulated by Purkinje cell stimulation, affecting mainly the activity in the theta and gamma bands, depending on ongoing behavior and their precise site in the cerebellar hemispheres.     

Peripheral sensory stimulation elicits global slow waves by recruiting somatosensory cortex bilaterally

Slow waves (SWs) are globally propagating, low-frequency (0.5- to 4-Hz) oscillations that are prominent during sleep and anesthesia. SWs are essential to neural plasticity and memory. However, much remains unknown about the mechanisms coordinating SW propagation at the macroscale. To assess SWs in the context of macroscale networks, we recorded cortical activity in awake and ketamine/xylazine-anesthetized mice using widefield optical imaging with fluorescent calcium indicator GCaMP6f. We demonstrate that unilateral somatosensory stimulation evokes bilateral waves that travel across the cortex with state-dependent trajectories. Under anesthesia, we observe that rhythmic stimuli elicit globally resonant, front-to-back propagating SWs. Finally, photothrombotic lesions of S1 show that somatosensory-evoked global SWs depend on bilateral recruitment of homotopic primary somatosensory cortices. Specifically, unilateral lesions of S1 disrupt somatosensory-evoked global SW initiation from either hemisphere, while spontaneous SWs are largely unchanged. These results show that evoked SWs may be triggered by bilateral activation of specific, homotopically connected cortical networks.

Slow waves (SWs) are the predominant cortical rhythm during nonrapid eye movement sleep and anesthesia (1, 2). Since they were first characterized three decades ago, SWs have been linked to a variety of brain functions, including memory consolidation (3–6), homeostatic synaptic plasticity (7), and grouping of other oscillatory events (8–10). Macroscopic recordings (scalp electroencephalogram [EEG]) have revealed that spontaneous global SWs occur approximately once per second (∼1 Hz) and propagate in a stereotypical front-to-back topography through the entire cortex (11). Local electrophysiology has demonstrated that virtually every cortical neuron participates in traveling SWs, exhibiting phase-locked alternation between depolarization (up-state) and hyperpolarization (down-state) (12, 13). During low arousal states, global SWs occur spontaneously but may also be evoked by peripheral sensory stimulation, as well as direct electromagnetic and optogenetic stimulation of the cortex (14–18). However, much remains unknown about the large-scale circuits supporting SW generation and propagation.Here, we investigate the role of the primary somatosensory cortex and its interhemispheric connections in the initiation and propagation of somatosensory-evoked SWs. To this end, we performed widefield optical imaging of cortical dynamics in awake and anesthetized mice expressing fluorescent calcium indicator GCaMP6f in pyramidal neurons. High-resolution mesoscopic imaging across the whole dorsal neocortex allows for a more-precise characterization of globally coherent waves of neural activity than conventional approaches for recording SWs (EEG or local electrophysiology). We demonstrate that unilateral somatosensory stimulation elicits bilateral waves that propagate in opposite directions depending on state (awake versus ketamine/xylazine anesthesia). Further, we show that the imposed rhythm of stimulation induces resonant activity locally in sensorimotor areas that remains focal in awake animals. In contrast, in anesthetized animals, rhythmic stimulation subsequently elicits SWs that spread globally in a front-to-back trajectory across the cortex. Finally, we use a photothrombotic stroke model to show that somatosensory-evoked global SWs depend on engagement of both ipsilateral and contralateral somatosensory cortex (S1). Unilateral photothrombosis of S1 disrupts global SWs evoked by peripheral stimulation of either hemisphere but spares the global spatiotemporal structure of spontaneous SWs outside of the perilesional area. These findings suggest a key role for bilateral recruitment of homotopically connected somatosensory cortices in initiating somatosensory-evoked global SWs and suggest potential mechanisms by which focal cortical injuries may influence global brain dynamics.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

Attention recruits frontal cortex in human infants

Young infants learn about the world by overtly shifting their attention to perceptually salient events. In adults, attention recruits several brain regions spanning the frontal and parietal lobes. However, it is unclear whether these regions are sufficiently mature in infancy to support attention and, more generally, how infant attention is supported by the brain. We used event-related functional magnetic resonance imaging (fMRI) in 24 sessions from 20 awake behaving infants 3 mo to 12 mo old while they performed a child-friendly attentional cuing task. A target was presented to either the left or right of the infant’s fixation, and offline gaze coding was used to measure the latency with which they saccaded to the target. To manipulate attention, a brief cue was presented before the target in three conditions: on the same side as the upcoming target (valid), on the other side (invalid), or on both sides (neutral). All infants were faster to look at the target on valid versus invalid trials, with valid faster than neutral and invalid slower than neutral, indicating that the cues effectively captured attention. We then compared the fMRI activity evoked by these trial types. Regions of adult attention networks activated more strongly for invalid than valid trials, particularly frontal regions. Neither behavioral nor neural effects varied by infant age within the first year, suggesting that these regions may function early in development to support the orienting of attention. Together, this furthers our mechanistic understanding of how the infant brain controls the allocation of attention.

Having an attention system that is capable of swiftly orienting to salient events (i.e., stimulus-driven attention) is essential for many behaviors. This is perhaps most true in infancy, during which exploration is thought to be critical (1) and attention allows infants to fully experience learning moments (2). The value of attention in early development might explain why infants are equipped with the capacity to flexibly allocate attention: They can saccade to onsets soon after birth (3), use cues to facilitate orienting (4, 5), and make predictions about upcoming events (6). Yet, how the infant brain supports attention remains a mystery.An extensive literature in adults could inform our understanding of the neural basis of stimulus-driven attention in infants. Regions collectively referred to as the frontoparietal network, including right temporal parietal junction (TPJ), superior parietal lobe (SPL), lateral occipital cortex (LOC), frontal eye fields (FEF), middle/inferior frontal gyrus (MFG/IFG), and pulvinar, have been implicated in the orienting of stimulus-driven and goal-directed attention (7–10). Other regions including the anterior cingulate cortex (ACC), insula, and basal ganglia, referred to as the cingulo-opercular or salience network (11), have been implicated in the maintenance and updating of task goals. However, the cingulo-opercular network is activated for stimulus-driven attention when the orienting is unexpected (12). Together, this suggests several functionally distinct regions that may be recruited for stimulus-driven attention across the age span.Yet, the regions that support stimulus-driven attention in adults are anatomically immature in infants (13–16), and functional connectivity between these regions, critical for supporting attention in adults (17, 18), undergoes rapid development in the first year of life (19–24). Indeed, these regions undergo functional changes late into adolescence (17, 18). Furthermore, the regions that support stimulus-driven attention in adults are also recruited for maintaining goals and volitionally directing attention based on them (i.e., goal-directed attention) (25–27). However, goal-directed attention is less developed than stimulus-driven attention in infants (28–31), suggesting immaturity in some parts of infant attention networks. These threads of evidence led to the proposal that some regions, like the MFG/IFG and ACC, are not sufficiently mature in infancy to support attention, and instead, the TPJ, SPL, and FEF are recruited (32). An alternative account suggests that the frontoparietal and cingulo-opercular networks are capable of functioning in early infancy (20, 33, 34), even if there is anatomical immaturity (35).Existing studies of the infant attention system have been inconclusive about the extent to which the infant brain recruits adult attention networks. Electroencephalography (EEG) with infants suggested that some neural signatures of attention (36, 37) and error processing (33, 38) are adult-like. However, EEG has insufficient spatial resolution to resolve which regions are supporting attention. Functional near-infrared spectroscopy (fNIRS) offers potentially greater resolution (6, 39), but is unable to localize activity beyond regions close to the scalp surface, including deeper, ventral, medial, and subcortical structures such as the ACC and basal ganglia. Moreover, many aspects of attention behavior develop throughout infancy (40), such as disengagement (41), inhibition (42), and goal-directed attention (28–31), raising the expectation that the underlying brain systems would also show age-dependent changes. Hence, it remains unclear how stimulus-driven attention is supported in the infant brain.We used functional magnetic resonance imaging (fMRI) and offline gaze coding to investigate the behavioral and neural basis of stimulus-driven attention in awake behaving infants under a year old. Among noninvasive techniques, fMRI is uniquely capable of resolving brain-wide, fine-grained attention processes. Recent innovations have made it possible to collect data from awake behaving infants (43–46). Using a developmental variant (4, 47) of the Posner cuing task (48), we simultaneously recorded gaze behavior and whole-brain fMRI activity to uncover the neural basis of attention in infants.     

Nonthermal and reversible control of neuronal signaling and behavior by midinfrared stimulation

Various neuromodulation approaches have been employed to alter neuronal spiking activity and thus regulate brain functions and alleviate neurological disorders. Infrared neural stimulation (INS) could be a potential approach for neuromodulation because it requires no tissue contact and possesses a high spatial resolution. However, the risk of overheating and an unclear mechanism hamper its application. Here we show that midinfrared stimulation (MIRS) with a specific wavelength exerts nonthermal, long-distance, and reversible modulatory effects on ion channel activity, neuronal signaling, and sensorimotor behavior. Patch-clamp recording from mouse neocortical pyramidal cells revealed that MIRS readily provides gain control over spiking activities, inhibiting spiking responses to weak inputs but enhancing those to strong inputs. MIRS also shortens action potential (AP) waveforms by accelerating its repolarization, through an increase in voltage-gated K⁺ (but not Na⁺) currents. Molecular dynamics simulations further revealed that MIRS-induced resonance vibration of –C=O bonds at the K⁺ channel ion selectivity filter contributes to the K⁺ current increase. Importantly, these effects are readily reversible and independent of temperature increase. At the behavioral level in larval zebrafish, MIRS modulates startle responses by sharply increasing the slope of the sensorimotor input–output curve. Therefore, MIRS represents a promising neuromodulation approach suitable for clinical application.

Many forms of neuromodulation have been used for the regulation of brain functions and the treatment of brain disorders. Some physical approaches, such as electrical, magnetic, and optical (electromagnetic; EM) stimulation could be employed to manipulate neural spiking activity and achieve neuromodulation. Among them, deep-brain electrical stimulation has become a gold standard treatment for advanced Parkinson’s disease; transcranial magnetic stimulation also generates electrical current in selected brain regions and has been used for mood regulation. In contrast, optical neural stimulation has not been used clinically, largely due to the risk of tissue damage by overheating and unclear mechanisms. Although optogenetic manipulation and stimulation avoid these problems and show cell specificity, the requirement of expression of exogenous genes hinders its use in humans (1).Optical infrared neural stimulation (INS) is emerging as an area of interest for neuromodulation and potential clinical application. INS utilizes brief light pulses to activate excitable cells or tissues in the illumination spot. Previous studies showed that INS could activate peripheral nerves (2), peripheral sensory systems (3, 4), and cardiac tissue (5). In the central nervous system (CNS), initial studies found that INS could evoke neural responses in rat thalamocortical slices in vitro (6) and regulate spiking activity in rodent somatosensory cortex (7) and nonhuman primate visual cortex in vivo (8). Because of its high spatial precision, focal INS has been recently applied to map brain connectomes (9). The underlying mechanism of INS, however, remains poorly understood. The predominant view is the transduction of EM energy to thermal heat (10, 11) will excite the cell, possibly due to heat-induced transmembrane capacitive charge (12), changes in ion channel activity (13), or cell damage (14). Since previous studies tended to choose infrared wavelengths with high water absorption for efficient heat generation, it remains unclear whether INS exerts nonthermal effects on ion channel and neuronal spiking activity.While most studies on infrared stimulation have been conducted at near-infrared wavelengths, whether midinfrared wavelengths can regulate neural function is unknown. Because the frequency of midinfrared light falls into the frequency range of chemical bond vibration (15–17), nonlinear resonances may occur within biomolecules (18–20), leading to dramatic changes in their conformation and function (21) and thus producing nonthermal effects on biological systems. Ion channel proteins distributed on cell membranes could be potential molecular targets for midinfrared light. Among them, voltage-gated Na⁺ and K⁺ channels play critical roles in regulating the initiation and propagation of the action potential (AP), an all-or-none digital signal of neurons (22, 23). They also control the voltage waveform of the AP and thus the size of the postsynaptic response, ensuring analog-mode communication between neurons (24–27). It is of interest to know whether midinfrared stimulation (MIRS) can cause conformational change in these channel proteins and consequently regulate neuronal signaling. Previous studies revealed a low absorption of light by water in the midinfrared region from 3.5 to 5.7 μm (28), which could be a potential wavelength range for neuromodulation. Therefore, in this study, we explored whether MIRS with a specific wavelength in this range could exert nonthermal modulatory effects on channel activity, neuronal signaling, and behavior.     

Spontaneous activity competes with externally evoked responses in sensory cortex

The interaction between spontaneous and externally evoked neuronal activity is fundamental for a functional brain. Increasing evidence suggests that bursts of high-power oscillations in the 15- to 30-Hz beta-band represent activation of internally generated events and mask perception of external cues. Yet demonstration of the effect of beta-power modulation on perception in real time is missing, and little is known about the underlying mechanism. Here, we used a closed-loop stimulus-intensity adjustment system based on online burst-occupancy analyses in rats involved in a forepaw vibrotactile detection task. We found that the masking influence of burst occupancy on perception can be counterbalanced in real time by adjusting the vibration amplitude. Offline analysis of firing rates (FRs) and local field potentials across cortical layers and frequency bands confirmed that beta-power in the somatosensory cortex anticorrelated with sensory evoked responses. Mechanistically, bursts in all bands were accompanied by transient synchronization of cell assemblies, but only beta-bursts were followed by a reduction of FR. Our closed loop approach reveals that spontaneous beta-bursts reflect a dynamic state that competes with external stimuli.

The brain is constantly active, even at resting states in the absence of external stimuli (1). Spontaneously active resting-state networks (RSNs) were found in memory, visual, auditory, tactile, and sensorimotor regions, with activity patterns similar to task-evoked responses (2, 3). Functional connectivity studies in humans suggest that a default network, spontaneously activated at resting states and deactivated upon increased cognitive demands, antagonizes a network involved in active attention to external sensory input (4–8). However, whether the activity in different networks is anticorrelated is under debate, and their antagonizing mechanisms and influence on local circuits remain unknown (9).Here, we utilized the occupancy of high-power bursts in the beta-band (15 to 30 Hz) of local field potentials (LFPs) as an indicator of spontaneous activity to investigate its influence on detection in real time. Several lines of evidence relate the RSN to beta-bursts. First, spontaneous correlated oscillatory activity in beta (termed “beta-connectome”) (10) was reported in anatomical regions corresponding to the RSN (11, 12). A recent study derived this beta-connectome from burst occupancy (10). Second, beta-oscillations are dominant during the resting state (13) and bursts are responsible for virtually all beta-band power modulation (14). Third, task-dependent desynchronization of beta was observed in the somatosensory (15, 16), visual (17), auditory (18), and motor (19) cortices, resembling RSN deactivation (4, 7). The task-dependent averaged power modulation was attributed to changes in burst rates in rodents, nonhuman primates, and humans (14, 20, 21). Fourth, the burst duration (50 to a few hundred milliseconds) (22) is similar to “packets” of neural activity, which are conceived as messages initiated in a particular cortical region and spread as a wave over the cortex. Most of these packets are generated spontaneously, and spontaneous and sensory-evoked packets are remarkably similar (23).We found that bursts in all bands indicate transient synchronization of flexible neuronal networks, but only beta-bursts were followed by a reduction in population firing rate (FR). High occupancies of beta-bursts predicted reduced detection, and this effect can be counterbalanced bidirectionally in real time by adjusting the stimulus intensity according to burst occupancy.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Improved bounds on entropy production in living systems

Living systems maintain or increase local order by working against the second law of thermodynamics. Thermodynamic consistency is restored as they consume free energy, thereby increasing the net entropy of their environment. Recently introduced estimators for the entropy production rate have provided major insights into the efficiency of important cellular processes. In experiments, however, many degrees of freedom typically remain hidden to the observer, and, in these cases, existing methods are not optimal. Here, by reformulating the problem within an optimization framework, we are able to infer improved bounds on the rate of entropy production from partial measurements of biological systems. Our approach yields provably optimal estimates given certain measurable transition statistics. In contrast to prevailing methods, the improved estimator reveals nonzero entropy production rates even when nonequilibrium processes appear time symmetric and therefore may pretend to obey detailed balance. We demonstrate the broad applicability of this framework by providing improved bounds on the energy consumption rates in a diverse range of biological systems including bacterial flagella motors, growing microtubules, and calcium oscillations within human embryonic kidney cells.

Thermodynamic laws place fundamental limits on the efficiency and fitness of living systems (1, 2). To maintain cellular order and perform essential biological functions such as sensing (3–6), signaling (7), replication (8, 9) or locomotion (10), organisms consume energy and dissipate heat. In doing so, they increase the entropy of their environment (2), in agreement with the second law of thermodynamics (11). Obtaining reliable estimates for the energy consumption and entropy production in living matter holds the key to understanding the physical boundaries (12–14) that constrain the range of theoretically and practically possible biological processes (3). Recent experimental (6, 15, 16) and theoretical (17–20) advances in the imaging and modeling of cellular and subcellular dynamics have provided groundbreaking insights into the thermodynamic efficiency of molecular motors (14, 21), biochemical signaling (16, 22, 23) and reaction (24) networks, and replication (9) and adaption (25) phenomena. Despite such major progress, however, it is also known that the currently available entropy production estimators (26, 27) can fail under experimentally relevant conditions, especially when only a small set of observables is experimentally accessible or nonequilibrium transport currents (28–30) vanish.To help overcome these limitations, we introduce here a generic optimization framework that can produce significantly improved bounds on the entropy production in living systems. We will prove that these bounds are optimal given certain measurable statistics. From a practical perspective, our method requires observations of only a few coarse-grained state variables of an otherwise hidden Markovian network. We demonstrate the practical usefulness by determining improved entropy production bounds for bacterial flagella motors (10, 31), growing microtubules (32, 33), and calcium oscillations (7, 34) in human embryonic kidney cells.Generally, entropy production rates can be estimated from the time series of stochastic observables (35). Thermal equilibrium systems obey the principle of detailed balance, which means that every forward trajectory is as likely to be observed as its time reversed counterpart, neutralizing the arrow of time (36). By contrast, living organisms operate far from equilibrium, which means that the balance between forward and reversed trajectories is broken and net fluxes may arise (1, 37–39). When all microscopic details of a nonequilibrium system are known, one can measure the rate of entropy production by comparing the likelihoods of forward and reversed trajectories in sufficiently large data samples (35, 36). However, in most if not all biophysical experiments, many degrees of freedom remain hidden to the observer, demanding methods (28, 40, 41) that do not require complete knowledge of the system. A powerful alternative is provided by thermodynamic uncertainty relations (TURs), which use the mean and variance of steady-state currents to bound entropy production rates (18, 19, 26, 42–48). Although highly useful when currents can be measured (44–47), or when the system can be externally manipulated (40, 49), these methods give, by construction, trivial zero bounds for current-free nonequilibrium systems, such as driven one-dimensional (1D) nonperiodic oscillators. In the absence of currents, potential asymmetries in the forward and reverse trajectories can still be exploited to bound the entropy production rate (29, 30, 50), but to our knowledge no existing method is capable of producing nonzero bounds when forward and reverse trajectories are statistically identical. Moreover, even though previous bounds can become tight in some cases (51), optimal entropy production estimators for nonequilibrium systems are in general unknown.To obtain bounds that are provably optimal under reasonable conditions on the available data, we reformulate the problem here within an optimization framework. Formally, we consider any steady-state Markovian dynamics for which only coarse-grained variables are observable, where these observables may appear non-Markovian. We then search over all possible underlying Markovian systems to identify the one which minimizes the entropy production rate while obeying the observed statistics. More specifically, our algorithmic implementation leverages information about successive transitions, allowing us to discover nonzero bounds on entropy production even when the coarse-grained statistics appear time symmetric. We demonstrate this for both synthetic test data and experimental data (52) for flagella motors. Subsequently, we consider the entropy production of microtubules (33), which slowly grow before rapidly shrinking in steady state, to show how refined coarse graining in space and time leads to improved bounds. The final application to calcium oscillations in human embryonic kidney cells (34) illustrates how external stimulation with drugs can increase entropy production.     

Invariant timescale hierarchy across the cortical somatosensory network

The ability of cortical networks to integrate information from different sources is essential for cognitive processes. On one hand, sensory areas exhibit fast dynamics often phase-locked to stimulation; on the other hand, frontal lobe areas with slow response latencies to stimuli must integrate and maintain information for longer periods. Thus, cortical areas may require different timescales depending on their functional role. Studying the cortical somatosensory network while monkeys discriminated between two vibrotactile stimulus patterns, we found that a hierarchical order could be established across cortical areas based on their intrinsic timescales. Further, even though subareas (areas 3b, 1, and 2) of the primary somatosensory (S1) cortex exhibit analogous firing rate responses, a clear differentiation was observed in their timescales. Importantly, we observed that this inherent timescale hierarchy was invariant between task contexts (demanding vs. nondemanding). Even if task context severely affected neural coding in cortical areas downstream to S1, their timescales remained unaffected. Moreover, we found that these time constants were invariant across neurons with different latencies or coding. Although neurons had completely different dynamics, they all exhibited comparable timescales within each cortical area. Our results suggest that this measure is demonstrative of an inherent characteristic of each cortical area, is not a dynamical feature of individual neurons, and does not depend on task demands.

There has been an increase in compelling evidence that cortical areas are diverse not only in their coding dynamics, but also in their structural and inherent features (1). This structural heterogeneity can be viewed as an anatomic feature (2), but it may also be a critical functional feature related to higher-order cortical computations, such as sensory processing, working memory, decision making, and perception (3). For instance, these cortical heterogeneities appear to be crucial for information flow across cortices (4, 5) and for proper brain function (6, 7). If this were the case, then these inherent heterogeneities could be used to establish a hierarchy across cortices. Recently, a hierarchy was found in mice, monkeys, and humans by estimating an intrinsic time constant from the dynamics of each cortical area (8–12). Neurons from sensory cortices exhibit much faster timescales than frontal lobe neurons. Additionally, this timescale hierarchy is parallel to the hierarchical order observed for the size of spatial receptive fields across visual and somatosensory pathways (13–15). In these cases, structural heterogeneities (timescales or receptive fields) yield hierarchies that also relate to function. While neurons from early sensory cortices can be phase-locked to stimulation, neurons from downstream areas are able to associate different signals to participate in working memory, decision-making, and perceptual processes (16, 17).Focusing on the somatosensory network, an anatomic hierarchy has been firmly established across cortical areas in primates and humans (2, 18, 19). When a stimulator moves perpendicular to the skin, cutaneous receptors are activated (20), giving rise to a signal that is conveyed by specific primary afferents to the spinal cord (21), then to the thalamus (22), and then up to the primary somatosensory cortex (S1) (13, 23). In S1, the sensory signal first arrives to area 3b (23). The next step in the somatosensory pathway is area 1. Historically, areas 3b and 1 were both considered parts of S1. Importantly, even when receptive fields increase from area 3b to area 1 (18, 24), their neuronal responses exhibit strikingly similar firing rate dynamics (13, 25, 26). This similarity makes it hard to distinguish these two areas from a coding perspective. The following cortical areas to be recruited are 2, 5, and 7b (27–29), which possess much larger receptive fields and longer response latencies but comparable phase-locking dynamics in the flutter range. The somatosensory inputs proceed to the secondary somatosensory cortex (S2), which shows a diversity of responses: ranging from pure sensory dynamics to more perceptual and categorical coding (13, 30, 31). In contrast to neurons from associative cortical areas, S2 neurons do not persistently code stimulus information during working memory periods (32). Associative areas from the frontal lobe—ventral, medial, and dorsal premotor cortices (VPC, MPC, and DPC) and prefrontal cortex (PFC)—exhibit heterogeneous responses associated with all processes involved during somatosensory tasks: sensory coding, working memory, comparisons, and decision-making (3, 32, 33).Here we used single-neuron activity recorded during a vibrotactile temporal pattern discrimination task (TPDT; Fig. 1 A and B) (34, 35) to study the inherent fluctuations during the basal period that precedes stimulus presentation. Recently, the timescale of these fluctuations was estimated with the decay of their autocorrelation (8–10, 36). We computed this autocorrelation decay across seven cortical areas that span different steps across the somatosensory network (Fig. 1C). Using this metric, we asked the following questions: what is the hierarchical order of these cortical areas in the somatosensory network? Further, is there any difference among the timescales of the subareas that compose S1? Additionally, we used a nondemanding variant of the TPDT as a control (light control task [LCT]), where the monkeys exhibit 100% performance (Fig. 1B). Coding dynamics during this control task are severely altered (34), so will autocorrelation and its suggested hierarchical order also be affected? Moreover, neurons exhibit heterogeneous coding responses in higher-order cortical areas, such as the DPC (31, 35); do timescales depend on the specific coding of neurons? In other words, in each cortical area, do the intrinsic timescales covary with the selectivity that each neuron displays, or are response selectivity and timescales independent dynamic traits?Open in a separate windowFig. 1.TPDT, performance, and recorded cortical areas. (A) Trials’ sequence of events. The mechanical probe is lowered (pd), indenting the glabrous skin of one fingertip of the right, restrained hand (500 µm); in response, the monkey places its left, free hand on an immovable key (kd). After a variable prestimulus period (from 2 to 4s), the probe vibrates for 1 s, generating one of two possible stimulus patterns (P1, either grouped [G] or extended [E]; mean frequency of 5 Hz). Note that in extended pattern E, pulses are delivered periodically. After a first delay (from 1 to 3 s) between P1 and P2, the second stimulus (P2) is delivered, again in either of the two possible patterns (P2, either G or E; 1 s duration); this is also called the comparison period. After a second 2-s delay (from 4 to 6 s) between the end of P2 and the probe up (pu), the monkey releases the key (ku) and presses, with its left free hand, either the lateral or the medial pushbutton (pb) to indicate whether the patterns were the same (P1 = P2) or different (P1 ≠ P2). (B) Performance for the whole TPDT (85%, gray; n_SES = 954 sessions), for each class (86% G-G [red], 83% G-E [orange], 84% E-G [green], 87% E-E [blue]) and for the entire LCT (100%, yellow; n_SES = 226 sessions). In the LTC, the same stimuli were delivered as in the TPDT, but the rewarding pushbutton press was visually guided. (C) Top (Left), lateral (Middle), and coronal (Right) views of the brain locations where single neurons were recorded. Cortical areas include areas 3b (green), 1 (blue), 2 (pink), 5 (violet), 7b (cyan), S2 (red), and DPC (orange). Recordings in S2 and DPC were made contralateral and ipsilateral to the stimulated fingertip.We confirmed that a hierarchical order can be established along the cortical somatosensory network based on autocorrelation decay. Remarkably, the historical primary somatosensory cortex can be divided into at least three different timescales of temporal integration: those of areas 3b, 1, and 2. Conversely, S2 and a frontal lobe area (DPC) exhibit comparable time constants. However, the specific autocorrelation values are much higher in the DPC. Stronger autocorrelation, which can be thought of as reverberation, may facilitate working memory in this area (17). Surprisingly, the hierarchy is preserved during the LCT. Although coding dynamics are severely affected in some areas during this nondemanding task (S2 and DPC), their autocorrelation functions are preserved. Furthermore, we separated neurons from area 1, S2, and DPC into subgroups with completely different coding and latencies. Notably, each subgroup of neurons displays a time constant comparable to its area’s whole population. We also compared hemispheres and obtained similar timescales for S2 and DPC. These results show strong evidence that time constants depict a hierarchical order across the somatosensory network, which is invariant under changes of context or coding dynamics and thus are inherent to each cortical processing stage.     

Selective filtering of excitatory inputs to nucleus accumbens by dopamine and serotonin

The detailed mechanisms by which dopamine (DA) and serotonin (5-HT) act in the nucleus accumbens (NAc) to influence motivated behaviors in distinct ways remain largely unknown. Here, we examined whether DA and 5-HT selectively modulate excitatory synaptic transmission in NAc medium spiny neurons in an input-specific manner. DA reduced excitatory postsynaptic currents (EPSCs) generated by paraventricular thalamus (PVT) inputs but not by ventral hippocampus (vHip), basolateral amygdala (BLA), or medial prefrontal cortex (mPFC) inputs. In contrast, 5-HT reduced EPSCs generated by inputs from all areas except the mPFC. Release of endogenous DA and 5-HT by methamphetamine (METH) and (±)3,4-methylenedioxymethamphetamine (MDMA), respectively, recapitulated these input-specific synaptic effects. Optogenetic inhibition of PVT inputs enhanced cocaine-conditioned place preference, whereas mPFC input inhibition reduced the enhancement of sociability elicited by MDMA. These findings suggest that the distinct, input-specific filtering of excitatory inputs in the NAc by DA and 5-HT contribute to their discrete behavioral effects.

The nucleus accumbens (NAc), a major node of classic mesolimbic reward circuitry, plays a critical role in a variety of adaptive and pathological motivated behaviors by integrating information carried by inputs from a broad range of brain areas with distinct, yet overlapping functions (1–6). Output from the NAc is provided by medium spiny neurons (MSNs), the activity of which strongly depends on excitatory inputs from these brain areas, most prominently the ventral hippocampus (vHip), periventricular thalamus (PVT), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) (3, 7–11). The NAc is also a behaviorally important target for two of the brain’s major neuromodulatory systems, dopamine (DA) and serotonin (5-HT) (1, 5, 6, 12–14). DA release in the NAc, whether caused by drugs of abuse or optogenetic stimulation, is powerfully reinforcing and plays a critical role in shaping operant responses (1, 4–6, 15–17). In contrast, unlike DA release, release of 5-HT in the NAc, generated either pharmacologically or optogenetically, is not acutely reinforcing but can powerfully influence sociability (18, 19).The robust differences in the behavioral consequences of DA and 5-HT release in the NAc suggest that these neuromodulators must influence MSN activity in, perhaps profoundly, different ways. Yet little is known about the detailed mechanisms by which these neuromodulators accomplish this task. Because of the importance of excitatory input in controlling MSN activity and the fact that both DA and 5-HT are well established to modulate excitatory synaptic transmission in the NAc (18, 20–23), we hypothesized that an important mechanism by which these neuromodulators might distinctly influence MSN activity is by differentially filtering incoming information from major input structures. Specifically, we hypothesized that DA and 5-HT would depress excitatory synaptic transmission in distinct, input-specific manners. Because of methodological limitations prior to the advent of optogenetics, virtually all previous work examining DA and 5-HT modulation of excitatory transmission in the NAc used bulk electrical stimulation of unknown inputs.Consistent with our hypothesis, exogenously applied DA and 5-HT, as well as release of endogenous DA and 5-HT, depressed excitatory synaptic transmission in distinct, input-specific manners. Input-specific optogenetic inhibition of excitatory inputs to the NAc revealed input-specific effects on conditioned place preference and sociability assays, which are affected by NAc release of DA and 5-HT, respectively. Together, these results provide evidence that the input-specific filtering of excitatory input from distinct brain regions contributes to the behavioral effects of DA and 5-HT release in the NAc and provides a foundation for further work elucidating the neural mechanisms by which modulation of NAc activity influences motivated behaviors.     

Multiscale photonic imaging of the native and implanted cochlea

The cochlea of our auditory system is an intricate structure deeply embedded in the temporal bone. Compared with other sensory organs such as the eye, the cochlea has remained poorly accessible for investigation, for example, by imaging. This limitation also concerns the further development of technology for restoring hearing in the case of cochlear dysfunction, which requires quantitative information on spatial dimensions and the sensorineural status of the cochlea. Here, we employed X-ray phase-contrast tomography and light-sheet fluorescence microscopy and their combination for multiscale and multimodal imaging of cochlear morphology in species that serve as established animal models for auditory research. We provide a systematic reference for morphological parameters relevant for cochlear implant development for rodent and nonhuman primate models. We simulate the spread of light from the emitters of the optical implants within the reconstructed nonhuman primate cochlea, which indicates a spatially narrow optogenetic excitation of spiral ganglion neurons.

In the case of profound sensorineural hearing impairment, cochlear implants (CIs) partially restore hearing by providing auditory information to the brain via electrical stimulation of the spiral ganglion neurons (SGNs). CIs enable speech understanding in the majority of the ∼700,000 users worldwide. However, current clinical CIs are limited by their wide current spread (1) resulting in poor coding of spectral information (2). Recently, cochlear optogenetics was proposed for stimulating the auditory nerve by light (3–10). As light can be better confined in space, the spread of excitation in the cochlea is lower (3, 9–11) and, hence, future optical CIs (oCIs) promise improved speech comprehension—especially in noisy background—as well as greater music appreciation.For the technical development of oCIs toward a future medical device, major efforts are currently being undertaken to devise multichannel optical stimulators for the cochlea (10, 12–17). As is the case for the electrodes of current CIs, future oCIs will place multiple stimulation channels, here microscale emitters, along the tonotopic axis of the cochlea. Further development of the oCIs requires precise estimates of parameters such as scala tympani size, optimal probe stiffness, and bending radius. Moreover, cochlear optogenetics employs gene transfer to the SGNs for which adeno-associated viruses (AAVs) seem promising candidate vectors (3–5, 8). AAV delivery has used injection of virus suspension via the round window (4, 8) or directly into Rosenthal’s canal (5, 9, 10). Therefore, the volumes of Rosenthal’s canal and the scalae tympani, vestibuli and media needed to be evaluated in order to estimate the required virus load for injection. Finally, the sensorineural status of the cochlea is highly relevant for future gene therapy and CI stimulation, and hence, quantitative imaging of sensory cells and neurons is an important objective.Here, we employed multiscale X-ray phase-contrast tomography (XPCT) and light-sheet fluorescence microscopy (LSFM) and provide an analysis of cochlear morphology for mice, rats, gerbils, guinea pigs, and marmosets. Each of these animal models offers unique advantages for auditory research. The mouse is readily available for genetic manipulation (e.g., ref. 18). Channelrhodopsin-expressing transgenic lines are available also for rats (19, 20) that offer a larger cochlea and can carry heavier implants than mice (21–24). Similarly, gerbils and guinea pigs are established rodent models for auditory research with larger-sized cochleae. Moreover, gerbils, which have low-frequency hearing more similar to humans, have already been employed for cochlear optogenetics (5, 9, 10, 24). Finally, we analyzed the cochlea of the common marmoset, as an established nonhuman primate model for auditory research (e.g., refs. 25, 26). Marmosets possess a rich vocalization repertoire and share a pitch perception mechanism with humans (27). Therefore, we compared cochlear insertion of newly designed oCIs with electrical cochlear implants (eCI) and modeled the optical spread of excitation in the marmoset cochlea.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

Ultrasound activates mechanosensitive TRAAK K+ channels through the lipid membrane

Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K⁺ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.

Manipulating cellular electrical activity is central to basic research and is clinically important for the treatment of neurological disorders including Parkinson’s disease, depression, epilepsy, and schizophrenia (1–4). Optogenetics, chemogenetics, deep brain stimulation (DBS), transcranial electrical stimulation, and transcranial magnetic stimulation are widely utilized neuromodulatory techniques, but each is associated with physical or biological limitations (5). Transcranial stimulation affords poor spatial resolution; deep brain stimulation and optogenetic manipulation typically require surgical implantation of stimulus delivery systems, and optogenetic and chemogenetic approaches necessitate genetic targeting of light- or small-molecule–responsive proteins.Ultrasound was first recognized to modulate cellular electrical activity almost a century ago, and ultrasonic neuromodulation has since been widely reported in the brain, peripheral nervous system, and heart of humans and model organisms (5–12). Ultrasonic neuromodulation has garnered increased attention for its advantageous physical properties. Ultrasound penetrates deeply through biological tissues and can be focused to sub-mm (3) volumes without transferring substantial energy to overlaying tissue, so it can be delivered noninvasively, for example, to deep structures in the brain through the skull. Notably, ultrasound generates excitatory and/or inhibitory effects depending on the system under study and stimulus paradigm (5, 13, 14).The mechanisms underlying the effects of ultrasound on excitable cells remain largely unknown (5, 13). Ultrasound can generate a combination of thermal and mechanical effects on targeted tissue (15, 16) in addition to potential off-target effects through the auditory system (17, 18). Thermal and cavitation effects, while productively harnessed to ablate tissue or transiently open the blood–brain barrier (19), require stimulation of higher power, frequency, and/or duration than typically utilized for neuromodulation (5). Intramembrane cavitation or compressive and expansive effects on lipid bilayers could generate nonselective currents that alter cellular electrical activity (5, 13). Alternatively, ultrasound could activate mechanosensitive ion channels through the deposition of acoustic radiation force that increases membrane tension or geometrically deforms the lipid bilayer (5, 15). Consistent with this notion, behavioral responses to ultrasound in Caenorhabditis elegans require mechanosensitive, but not thermosensitive, ion channels (20), and a number of mechanosensitive (and force-sensitive, but noncanonically mechanosensitive) ion channels have been implicated in cellular responses to ultrasound including two-pore domain K⁺ channels (K2Ps), Piezo1, MEC-4, TRPA1, MscL, and voltage-gated Na⁺ and Ca²⁺ channels (20–24, 25). Precisely how ultrasound impacts the activity of these channels is not known.To better understand mechanisms underlying ultrasonic neuromodulation, we investigated the effects of ultrasound on the mechanosensitive ion channel TRAAK (26, 27). K2P channels including TRAAK are responsible for so called “leak-type” currents because they approximate voltage- and time-independent K⁺-selective holes in the membrane, although more complex gating and regulation of K2P channels is increasingly appreciated (28, 29). TRAAK has a very low open probability in the absence of membrane tension and is robustly activated by force through the lipid bilayer (30–32). Mechanical activation of TRAAK involves conformational changes that prevent lipids from entering the channel to block K⁺ conduction (31). Gating conformational changes are associated with shape changes that expand the channel and make it more cylindrical in the membrane plane upon opening. These shape changes are energetically favored in the presence of membrane tension, resulting in a tension-dependent energy difference between states that favors channel opening (31). TRAAK is expressed in neurons and has been localized exclusively to nodes of Ranvier, the excitable action potential propagating regions of myelinated axons (33, 34). TRAAK is found in most (∼80%) myelinated nerve fibers in both the central and peripheral nervous systems, where it accounts for ∼25% of basal nodal K⁺ currents. As in heterologous systems, mechanical stimulation robustly activates nodal TRAAK. TRAAK is functionally important for setting the resting potential and maintaining voltage-gated Na⁺ channel availability for spiking in nodes; loss of TRAAK function impairs high-speed and high-frequency nerve conduction (33, 34). Changes in TRAAK activity therefore appear well poised to widely impact neuronal excitability.We find that low-intensity and short-duration ultrasound rapidly and robustly activates TRAAK channels. Activation is observed in patches from TRAAK-expressing Xenopus oocytes, in patches containing purified channels reconstituted into lipid membranes, and in TRAAK-expressing mouse cortical neurons. Single-channel recordings reveal that canonical mechanical and ultrasonic activation are accomplished through a shared mechanism. We conclude that ultrasound activates TRAAK through the lipid membrane, likely by increasing membrane tension to promote channel opening. This work demonstrates direct mechanical activation of an ion channel by ultrasound using purified and reconstituted components, is consistent with endogenous mechanosensitive channel activity underlying physiological effects of ultrasound, and provides a framework for the development of exogenously expressed sonogenetic tools for ultrasonic control of neural activity.