A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

Estimation of skeletal kinematics in freely moving rodents.

Forming a complete picture of the relationship between neural activity and skeletal kinematics requires quantification of skeletal joint biomechanics during free behavior; however, without detailed knowledge of the underlying skeletal motion, inferring limb kinematics using surface-tracking approaches is difficult, especially for animals where the relationship between the surface and underlying skeleton changes during motion. Here we developed a videography-based method enabling detailed three-dimensional kinematic quantification of an anatomically defined skeleton in untethered freely behaving rats and mice. This skeleton-based model was constrained using anatomical principles and joint motion limits and provided skeletal pose estimates for a range of body sizes, even when limbs were occluded. Model-inferred limb positions and joint kinematics during gait and gap-crossing behaviors were verified by direct measurement of either limb placement or limb kinematics using inertial measurement units. Together we show that complex decision-making behaviors can be accurately reconstructed at the level of skeletal kinematics using our anatomically constrained model.

Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats.

We designed a head-mounted three-photon microscope for imaging deep cortical layer neuronal activity in a freely moving rat. Delivery of high-energy excitation pulses at 1,320 nm required both a hollow-core fiber whose transmission properties did not change with fiber movement and dispersion compensation. These developments enabled imaging at >1.1 mm below the cortical surface and stable imaging of layer 5 neuronal activity for >1 h in freely moving rats performing a range of behaviors.

Effectiveness of a Multi-component Delirium Prevention Program Implemented on General Medicine Hospital Units: an Interrupted Time Series Analysis.

BACKGROUND: Delirium is among the most prevalent harmful events in hospitals that is associated with an elevated risk for severe outcomes such as functional decline, falls, longer length of stay, and increased mortality. OBJECTIVE: To evaluate the impact of the implementation of a multi-component delirium program on the prevalence of delirium and the incidence of falls among patients staying on general medicine inpatient hospital units. DESIGN: A pre-post intervention study using retrospective chart abstraction and interrupted time series analysis. COHORT: Patients were selected from adult patients that stayed at least 1 day on one of the five general medicine units in a large community hospital in Ontario, Canada. A total of 16 random samples of 50 patients per month for 8 consecutive months pre-intervention (October 2017 to May 2018) and 8 months post intervention (January 2019 to August 2019) were selected for a total of 800 patients. There were no exclusion criteria. INTERVENTION: The delirium program included multiple components: education of staff and hospital leadership, twice per day bed-side screen for delirium, non-pharmacological and pharmacological prevention, and intervention strategies and a delirium consultation team. MEASUREMENT: Delirium prevalence was assessed using the evidence-based delirium chart abstraction method, CHART-del. Demographic data as well as fall incidence were also collected. RESULT: Our evaluation showed that the implementation of a multicomponent delirium program led to a reduction in delirium prevalence and fall incidences. The reduction in both delirium and falls was the largest for patients in the ages between 72 and 83 years old and varied across inpatient units. CONCLUSION: A multi-component delirium program to improve the prevention, recognition, and management of delirium reduces the prevalence of delirium and fall incidence among patients in general medicine units.

Linking catchment structural units (CSUs) with water quality: Implications for ambient monitoring network design and data interpretation.

Climate induced changes in runoff regimes and ongoing anthropogenic modification of land use and land cover (LULC) are shifting ambient water quality signals worldwide. Modulation of these signals by the physical catchment structure over different scales adds complexity to interpreting and analyzing measured data. Further bias may be introduced where monitoring networks are not representative of the structure of catchments in a given region. Here, we present a new environmental regionalization method to assess the representativeness of water quality monitoring (WQM) networks and to identify key structural drivers linked to water quality signals. Unique numerical codes were generated at the pixel level to provide wall-to-wall coverage of key Catchment Structural Units (CSUs) based on LULC, surficial geology, wetlands and slope. CSU codes were generated for all tributary (AT) catchments >20 km2 in Southern Alberta (n = 289), Canada, to determine the representativeness of an existing WQM network (54 tributary catchments) and to assess the explanatory power of CSUs with respect to water quality signals. Cluster analysis (CA) and multi-dimensional scaling (MDS) on the percent area of CSUs in the AT catchments identified six primary structural clusters in Southern Alberta. A clear gradient in catchment structure was evident progressing downstream from the Rocky Mountain headwaters through the foothills and prairie/plains region. Montane and grassland regions were found to be potentially under-represented by the current WQM program whereas catchments dominated by agriculture were likely over-represented. The disproportionate impact of specific CSU combinations on water quality was illustrated where the CA and MDS analyses indicated that even small percentages of urban areas and badland type topography results in elevated concentrations of total recoverable metals, nutrients and major ions. The application of the CSU approach in Southern Alberta demonstrates its value as an alternative method to assess and/or redesign existing WQM networks and to link water quality data to the structural composition of catchments. The general availability of the required data to generate CSUs provides universal potential for the approach to help assess other WQM programs and to contextualize data records. Applying the CSU approach when developing new ambient WQM networks can also help reduce the potential of over-monitoring similarly structured catchments as well as ensuring that all structural classes are represented by the data being generated.

Rats maintain an overhead binocular field at the expense of constant fusion.

Fusing left and right eye images into a single view is dependent on precise ocular alignment, which relies on coordinated eye movements. During movements of the head this alignment is maintained by numerous reflexes. Although rodents share with other mammals the key components of eye movement control, the coordination of eye movements in freely moving rodents is unknown. Here we show that movements of the two eyes in freely moving rats differ fundamentally from the precisely controlled eye movements used by other mammals to maintain continuous binocular fusion. The observed eye movements serve to keep the visual fields of the two eyes continuously overlapping above the animal during free movement, but not continuously aligned. Overhead visual stimuli presented to rats freely exploring an open arena evoke an immediate shelter-seeking behaviour, but are ineffective when presented beside the arena. We suggest that continuously overlapping visual fields overhead would be of evolutionary benefit for predator detection by minimizing blind spots.

Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites.

Cortical inhibitory interneurons (INs) are subdivided into a variety of morphologically and functionally specialized cell types. How the respective specific properties translate into mechanisms that regulate sensory-evoked responses of pyramidal neurons (PNs) remains unknown. Here, we investigated how INs located in cortical layer 1 (L1) of rat barrel cortex affect whisker-evoked responses of L2 PNs. To do so we combined in vivo electrophysiology and morphological reconstructions with computational modeling. We show that whisker-evoked membrane depolarization in L2 PNs arises from highly specialized spatiotemporal synaptic input patterns. Temporally L1 INs and L2-5 PNs provide near synchronous synaptic input. Spatially synaptic contacts from L1 INs target distal apical tuft dendrites, whereas PNs primarily innervate basal and proximal apical dendrites. Simulations of such constrained synaptic input patterns predicted that inactivation of L1 INs increases trial-to-trial variability of whisker-evoked responses in L2 PNs. The in silico predictions were confirmed in vivo by L1-specific pharmacological manipulations. We present a mechanism-consistent with the theory of distal dendritic shunting-that can regulate the robustness of sensory-evoked responses in PNs without affecting response amplitude or latency.

Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A.

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase. We found that a column contains ~2,200 INs (11.5% of ~19,000 neurons), almost a factor of 2 less than previously estimated. The density of GABAergic neurons was inhomogeneous between layers, with peaks in the upper third of L2/3 and in L5A. IN density therefore defines a distinct layer 2 in the sensory neocortex. In addition, immunohistochemical markers of IN subtypes were layer-specific. The "hot zones" of inhibition in L2 and L5A match the reported low stimulus-evoked spiking rates of excitatory neurons in these layers, suggesting that these inhibitory hot zones substantially suppress activity in the neocortex.

Nuclear receptor-binding protein 1: a novel tumour suppressor and pseudokinase.

Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.

Imaging in vivo: watching the brain in action.

The appeal of in vivo cellular imaging to any neuroscientist is not hard to understand: it is almost impossible to isolate individual neurons while keeping them and their complex interactions with surrounding tissue intact. These interactions lead to the complex network dynamics that underlie neural computation which, in turn, forms the basis of cognition, perception and consciousness. In vivo imaging allows the study of both form and function in reasonably intact preparations, often with subcellular spatial resolution, a time resolution of milliseconds and a purview of months. Recently, the limits of what can be achieved in vivo have been pushed into terrain that was previously only accessible in vitro, due to advances in both physical-imaging technology and the design of molecular contrast agents.

Steric effects in peptide and protein exchange with activated disulfides.

Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (ß-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

Chasing cortical behavior: designing multiphoton microscopes for imaging neuronal populations in freely moving rodents.

Imaging in the freely moving animal gives unparalleled access to circuit activity as the animal interacts with its environment in a self-guided way. Over the past few years, new imaging technologies have enabled the interrogation of neuronal populations located at any depth of the cortex in freely moving mice while preserving the animal's behavioral repertoire. This commentary gives an updated overview of the recent advances that have enabled the link between behavior and the underlying neuronal activity to be explored.

Improving the Prevention, Detection, and Management of Delirium in Adult Inpatients; an Interprofessional Consultative Team Pilot Project.

Delirium is a common, often preventable fluctuating state of cognition associated with increased morbidity and mortality. This report describes the implementation of an interprofessional consultative Delirium Team formed to improve the prevention, detection, and management of delirium in a community hospital. Team members consulted refered inpatients with delirium to establish a care plan and provide recommendations for pharmacological and non-pharmacological management. The team also offered delirium-related education to unit staff, patients, and caregivers. Consultations were initially completed by the team Nurse Practitioner or Occupational Therapist, and complex patients were discussed with the team Geriatrician and Psychiatrist at rounds to optimize specialist input. Of the 160 patients managed by the team over the 8-month study period, two-thirds of referred patients did not require specialist consultation for their delirium management. Strategies most often recommended by experts for managing delirium were related to medical management, social/cognitive engagement, and functional mobility. Two-thirds of all recommendations made by the team were implemented. Barriers and facilitators to implementation and improving unit staff adherence are further described. The consultative Delirium Team is a promising model that should be further explored for managing an aging population in a capacity-limited medical system.

Visually evoked activity in cortical cells imaged in freely moving animals.

We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca(2+) transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 trials per imaging session, even as the animal was running at velocities of up to 0.6 m/s. Neuronal Ca(2+) transients were readily detected, and responses to various static visual stimuli were observed during free movement on a running track. Neuronal activity was sparse and increased when the animal swept its gaze across a visual stimulus. Neurons showing preferential activation by specific stimuli were observed in freely moving animals. These results demonstrate that the multiphoton fiberscope is suitable for functional imaging in awake and freely moving animals.

Population imaging of ongoing neuronal activity in the visual cortex of awake rats.

It is unclear how the complex spatiotemporal organization of ongoing cortical neuronal activity recorded in anesthetized animals relates to the awake animal. We therefore used two-photon population calcium imaging in awake and subsequently anesthetized rats to follow action potential firing in populations of neurons across brain states, and examined how single neurons contributed to population activity. Firing rates and spike bursting in awake rats were higher, and pair-wise correlations were lower, compared with anesthetized rats. Anesthesia modulated population-wide synchronization and the relationship between firing rate and correlation. Overall, brain activity during wakefulness cannot be inferred using anesthesia.

Effectiveness of Interprofessional Consultation-Based Interventions for Delirium: A Scoping Review.

BACKGROUND: Interprofessional geriatric consultation teams and multicomponent interventions are established models for delirium care. They are combined in interprofessional consultative delirium team interventions; however, insight into this novel approach is lacking. OBJECTIVE: To describe the effectiveness and core components of consultation-based interventions for delirium. METHOD: Ovid MEDLINE, EMBASE, PsycINFO, CINAHL, and ProQuest. Data on core intervention components, outcomes, facilitators, and barriers were extracted. RESULTS: 10 studies were included. Core intervention components were systematic delirium screening, ongoing consultation, implementation of non-pharmacologic and pharmacological interventions, and staff education. Of the included studies, 1/6 found a significant reduction in delirium incidence, 1/2 a reduction in delirium duration, and 2/3 found a reduction in falls. Facilitators and barriers to implementation were discussed. CONCLUSION: There was consistency in team structure and core components, however intervention operationalization and effectiveness varied widely. There is some evidence that this model is effective for reducing delirium and its sequelae.

Measurement of arbitrary scan patterns for correction of imaging distortions in laser scanning microscopy.

Laser scanning microscopy requires beam steering through relay and focusing optics at sub-micron precision. In light-weight mobile systems, such as head mounted multiphoton microscopes, distortion and imaging plane curvature management is unpractical due to the complexity of required optic compensation. Thus, the resulting scan pattern limits anatomical fidelity and decreases analysis algorithm efficiency. Here, we present a technique that reconstructs the three-dimensional scan path only requiring translation of a simple fluorescent test probe. Our method is applicable to any type of scanning instrument with sectioning capabilities without prior assumptions regarding origin of imaging deviations. Further, we demonstrate that the obtained scan pattern allows analysis of these errors, and allows to restore anatomical accuracy relevant for complementary methods such as motion correction, further enhancing spatial registration and feature extraction.

Connectomic analysis of thalamus-driven disinhibition in cortical layer 4.

Sensory signals are transmitted via the thalamus primarily to layer 4 (L4) of the primary sensory cortices. While information about average neuronal connectivity in L4 is available, its detailed higher-order circuit structure is not known. Here, we used three-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in L4. We find that thalamic input drives a subset of interneurons with high specificity, which in turn target excitatory neurons with subtype specificity. These interneurons create a directed disinhibitory network directly driven by the thalamic input. Neuronal activity recordings show that strong synchronous sensory activation yields about 1.5-fold stronger activation of star pyramidal cells than spiny stellates, in line with differential windows of opportunity for activation of excitatory neurons in the thalamus-driven disinhibitory circuit model. With this, we have identified a high degree of specialization of the microcircuitry in L4 of the primary sensory cortex.

Visual pursuit behavior in mice maintains the pursued prey on the retinal region with least optic flow.

Mice have a large visual field that is constantly stabilized by vestibular ocular reflex (VOR) driven eye rotations that counter head-rotations. While maintaining their extensive visual coverage is advantageous for predator detection, mice also track and capture prey using vision. However, in the freely moving animal quantifying object location in the field of view is challenging. Here, we developed a method to digitally reconstruct and quantify the visual scene of freely moving mice performing a visually based prey capture task. By isolating the visual sense and combining a mouse eye optic model with the head and eye rotations, the detailed reconstruction of the digital environment and retinal features were projected onto the corneal surface for comparison, and updated throughout the behavior. By quantifying the spatial location of objects in the visual scene and their motion throughout the behavior, we show that the prey image consistently falls within a small area of the VOR-stabilized visual field. This functional focus coincides with the region of minimal optic flow within the visual field and consequently area of minimal motion-induced image-blur, as during pursuit mice ran directly toward the prey. The functional focus lies in the upper-temporal part of the retina and coincides with the reported high density-region of Alpha-ON sustained retinal ganglion cells.

Mice have a lot to keep an eye on. To survive, they need to dodge predators looming on land and from the skies, while also hunting down the small insects that are part of their diet. To do this, they are helped by their large panoramic field of vision, which stretches from behind and over their heads to below their snouts. To stabilize their gaze when they are on the prowl, mice reflexively move their eyes to counter the movement of their head: in fact, they are unable to move their eyes independently. This raises the question: what part of their large visual field of view do these rodents use when tracking a prey, and to what advantage? This is difficult to investigate, since it requires simultaneously measuring the eye and head movements of mice as they chase and capture insects. In response, Holmgren, Stahr et al. developed a new technique to record the precise eye positions, head rotations and prey location of mice hunting crickets in surroundings that were fully digitized at high resolution. Combining this information allowed the team to mathematically recreate what mice would see as they chased the insects, and to assess what part of their large visual field they were using. This revealed that, once a cricket had entered any part of the mice's large field of view, the rodents shifted their head ­ but not their eyes ­ to bring the prey into both eye views, and then ran directly at it. If the insect escaped, the mice repeated that behavior. During the pursuit, the cricket's position was mainly held in a small area of the mouse's view that corresponds to a specialized region in the eye which is thought to help track objects. This region also allowed the least motion-induced image blur when the animals were running forward. The approach developed by Holmgren, Stahr et al. gives a direct insight into what animals see when they hunt, and how this constantly changing view ties to what happens in the eyes. This method could be applied to other species, ushering in a new wave of tools to explore what freely moving animals see, and the relationship between behaviour and neural circuitry.