Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

A neural circuit state change underlying skilled movements.

In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.

Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks.

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task.

Throughout mammalian neocortex, layer 5 pyramidal (L5) cells project via the pons to a vast number of cerebellar granule cells (GrCs), forming a fundamental pathway. Yet, it is unknown how neuronal dynamics are transformed through the L5→GrC pathway. Here, by directly comparing premotor L5 and GrC activity during a forelimb movement task using dual-site two-photon Ca2+ imaging, we found that in expert mice, L5 and GrC dynamics were highly similar. L5 cells and GrCs shared a common set of task-encoding activity patterns, possessed similar diversity of responses, and exhibited high correlations comparable to local correlations among L5 cells. Chronic imaging revealed that these dynamics co-emerged in cortex and cerebellum over learning: as behavioral performance improved, initially dissimilar L5 cells and GrCs converged onto a shared, low-dimensional, task-encoding set of neural activity patterns. Thus, a key function of cortico-cerebellar communication is the propagation of shared dynamics that emerge during learning.

Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications.

For nearly 50 years the proximal tubule (PT) has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes. Several genetic diseases, nonselective PT cell defects, chronic kidney disease (CKD), and acute PT injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PT injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism, and transcytosis are being reexamined with the use of techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multireceptor complex that mediates albumin binding and uptake and directs proteins for lysosomal degradation after endocytosis. Albumin transcytosis is mediated by a pH-dependent binding affinity to the neonatal Fc receptor (FcRn) in the endosomal compartments. This reclamation pathway rescues albumin from urinary losses and cellular catabolism, extending its serum half-life. Albumin that has been altered by oxidation, glycation, or carbamylation or because of other bound ligands that do not bind to FcRn traffics to the lysosome. This molecular sorting mechanism reclaims physiological albumin and eliminates potentially toxic albumin. The clinical importance of PT albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussion of genetic disorders and therapeutic considerations.

Cognitive-Affective Functions of the Cerebellum.

The cerebellum, traditionally associated with motor coordination and balance, also plays a crucial role in various aspects of higher-order function and dysfunction. Emerging research has shed light on the cerebellum's broader contributions to cognitive, emotional, and reward processes. The cerebellum's influence on autonomic function further highlights its significance in regulating motivational and emotional states. Perturbations in cerebellar development and function have been implicated in various neurodevelopmental disorders, including autism spectrum disorder and attention deficit hyperactivity disorder. An increasing appreciation for neuropsychiatric symptoms that arise from cerebellar dysfunction underscores the importance of elucidating the circuit mechanisms that underlie complex interactions between the cerebellum and other brain regions for a comprehensive understanding of complex behavior. By briefly discussing new advances in mapping cerebellar function in affective, cognitive, autonomic, and social processing and reviewing the role of the cerebellum in neuropathology beyond the motor domain, this Mini-Symposium review aims to provide a broad perspective of cerebellar intersections with the limbic brain in health and disease.

The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells.

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Glycosylation of a key cubilin Asn residue results in reduced binding to albumin.

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.

Cerebellar granule cells encode the expectation of reward.

The human brain contains approximately 60 billion cerebellar granule cells, which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes. Although evidence suggests a role for the cerebellum in cognition, granule cells are known to encode only sensory and motor context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.

Kilohertz two-photon brain imaging in awake mice.

Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

Slower gait speed increases risk of falling in older adults with depression and cognitive complaints.

Slowed gait is one of the strongest predictors of fall risk in older adults. The present study investigated whether gait speed mediated the relationship between depression and fall history in 147 older adults presenting to a memory clinic for cognitive complaints. Depression, cognitive status, gait speed, and number of falls within the last year were the primary measures. Results revealed fallers, relative to non-fallers, had slower gait speed and higher depression scores. As hypothesized, analyses using the PROCESS macro found that gait mediated the relationship between depression and fall history. Additionally, the combination of depression and mild cognitive impairments (MCI) associated with a significantly greater likelihood of falling. Our findings indicate that combined depression and MCI have additive effects on fall risk, likely through the destabilizing effect of slowed gait on balance. Better understanding the underlying pathophysiology involved in MCI and depression-related gait disturbances may lead to improved intervention targets for fall risk prevention.

Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Evaluation of Endoscopic Iliopsoas Tenotomy for Treatment of Iliopsoas Impingement After Total Hip Arthroplasty.

BACKGROUND: Iliopsoas impingement after total hip arthroplasty (THA) occurs in up to 4.3% of patients resulting in functional groin pain. Operative treatment historically has included open iliopsoas tenotomy or acetabulum revision. We present a large single surgeon series of patients treated with endoscopic iliopsoas tenotomy for iliopsoas impingement after THA to evaluate the effectiveness and risks. METHODS: A consecutive series of 60 patients with iliopsoas impingement after THA treated with endoscopic iliopsoas tenotomy was retrospectively evaluated. Outcomes assessed were resolution of pain, change in Hip Outcome Score (HOS), and complications. Radiographs were reviewed by a musculoskeletal radiologist to evaluate component positioning and to compare with a control cohort. RESULTS: At last follow-up (mean 5.5 months), 93.3% of patients had resolution of pain. The HOS activities of daily living (ADL) subscale mean was 57.5 (range 10.9-89.3, standard deviation [SD] 18.8) preoperatively and 71.6 (range 14.1-100, SD 26.1) postoperatively (P = .005). The HOS sports subscale mean was 37.3 (range 0-83.3, SD 24.0) preoperatively and 58.1 (range 0-100, SD 33.2) postoperatively (P = .002). One complication was reported, a postoperative hematoma managed conservatively. Body mass index and increased offset were associated with iliopsoas symptoms after THA in this series. CONCLUSION: Endoscopic iliopsoas tenotomy after THA had a 93.3% resolution of pain, clinically important improvements in HOS, and low rate of complications. Endoscopic tenotomy should be considered as a treatment option in patients with iliopsoas impingement after THA.

Improving Sensitivity to Detect Mild Cognitive Impairment: Cognitive Load Dual-Task Gait Speed Assessment.

OBJECTIVES: Longitudinal research indicates that cognitive load dual-task gait assessment is predictive of cognitive decline and thus might provide a sensitive measure to screen for mild cognitive impairment (MCI). However, research among older adults being clinically evaluated for cognitive concerns, a defining feature of MCI, is lacking. The present study investigated the effect of performing a cognitive task on normal walking speed in patients presenting to a memory clinic with cognitive complaints. METHODS: Sixty-one patients with a mean age of 68 years underwent comprehensive neuropsychological testing, clinical interview, and gait speed (simple- and dual-task conditions) assessments. Thirty-four of the 61 patients met criteria for MCI. RESULTS: Repeated measure analyses of covariance revealed that greater age and MCI both significantly associated with slower gait speed, ps<.05. Follow-up analysis indicated that the MCI group had significantly slower dual-task gait speed but did not differ in simple-gait speed. Multivariate linear regression across groups found that executive attention performance accounted for 27.4% of the variance in dual-task gait speed beyond relevant demographic and health risk factors. CONCLUSIONS: The present study increases the external validity of dual-task gait assessment of MCI. Differences in dual-task gait speed appears to be largely attributable to executive attention processes. These findings have clinical implications as they demonstrate expected patterns of gait-brain behavior relationships in response to a cognitive dual task within a clinically representative population. Cognitive load dual-task gait assessment may provide a cost efficient and sensitive measure to detect older adults at high risk of a dementia disorder. (JINS, 2017, 23, 493-501).

Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level.

Mechanism of increased clearance of glycated albumin by proximal tubule cells.

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins.

The proximal tubule and albuminuria: really!

Recent data highlight the role of the proximal tubule (PT) in reabsorbing, processing, and transcytosing urinary albumin from the glomerular filtrate. Innovative techniques and approaches have provided exciting insights into these processes, and numerous investigators have shown that selective PT cell defects lead to significant albuminuria, even reaching nephrotic range in animal models. Thus, the mechanisms of albumin reabsorption and transcytosis are undergoing intense study. Working in concert with megalin and cubilin, a nonselective multireceptor complex that predominantly directs proteins for lysosomal degradation, the neonatal Fc receptor (FcRn) located at the brush border of the apical membrane has been implicated as the "receptor" mediating albumin transcytosis. The FcRn pathway facilitates reabsorption and mediates transcytosis by its pH-dependent binding affinity in endosomal compartments. This also allows for selective albumin sorting within the PT cell. This reclamation pathway minimizes urinary losses and catabolism of albumin, thus prolonging its serum half-life. It may also serve as a molecular sorter to preserve and reclaim normal albumin while allowing "altered" albumin to be catabolized via lysosomal pathways. Here, we critically review the data supporting this novel mechanism.

ARHGAP21 protein, a new partner of α-tubulin involved in cell-cell adhesion formation and essential for epithelial-mesenchymal transition.

Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.

High-global warming potential F-gas emissions in California: comparison of ambient-based versus inventory-based emission estimates, and implications of refined estimates.

To provide information for greenhouse gas reduction policies, the California Air Resources Board (CARB) inventories annual emissions of high-global-warming potential (GWP) fluorinated gases, the fastest growing sector of greenhouse gas (GHG) emissions globally. Baseline 2008 F-gas emissions estimates for selected chlorofluorocarbons (CFC-12), hydrochlorofluorocarbons (HCFC-22), and hydrofluorocarbons (HFC-134a) made with an inventory-based methodology were compared to emissions estimates made by ambient-based measurements. Significant discrepancies were found, with the inventory-based emissions methodology resulting in a systematic 42% under-estimation of CFC-12 emissions from older refrigeration equipment and older vehicles, and a systematic 114% overestimation of emissions for HFC-134a, a refrigerant substitute for phased-out CFCs. Initial, inventory-based estimates for all F-gas emissions had assumed that equipment is no longer in service once it reaches its average lifetime of use. Revised emission estimates using improved models for equipment age at end-of-life, inventories, and leak rates specific to California resulted in F-gas emissions estimates in closer agreement to ambient-based measurements. The discrepancies between inventory-based estimates and ambient-based measurements were reduced from -42% to -6% for CFC-12, and from +114% to +9% for HFC-134a.

Clinical endocannabinoid deficiency (CECD) revisited: can this concept explain the therapeutic benefits of cannabis in migraine, fibromyalgia, irritable bowel syndrome and other treatment-resistant conditions?

OBJECTIVES: Ethan B. Russo's paper of December 1, 2003 explored the concept of a clinical endocannabinoid deficiency (CECD) underlying the pathophysiology of migraine, fibromyalgia, irritable bowel syndrome and other functional conditions alleviated by clinical cannabis. METHODS: Available literature was reviewed, including searches via the National Library of medicine database and other sources. RESULTS: A review of the literature indicates that significant progress has been made since Dr. Ethan B. Russo's landmark paper, just ten years ago (February 2, 2004). Investigation at that time suggested that cannabinoids can block spinal, peripheral and gastrointestional mechanisms that promote pain in headache, fibromyalgia, irritable bowel syndrome and muscle spasm. CONCLUSION: Subsequent research has confirmed that underlying endocannabinoid deficiencies indeed play a role in migraine, fibromyalgia, irritable bowel syndrome and a growing list of other medical conditions. Clinical experience is bearing this out. Further research and especially, clinical trials will further demonstrate the usefulness of medical cannabis. As legal barriers fall and scientific bias fades this will become more apparent.