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.

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.

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.

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.

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.

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.

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.

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.

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.

Severe western Canadian wildfire affects water quality even at large basin scales.

Wildfires can have severe and lasting impacts on the water quality of aquatic ecosystems. However, our understanding of these impacts is founded primarily from studies of small watersheds with well-connected runoff regimes. Despite the predominance of large, low-relief rivers across the fire-prone Boreal forest, it is unclear to what extent and duration wildfire-related material (e.g., ash) can be observed within these systems that typically buffer upstream disturbance signals. Following the devastating 2016 Fort McMurray wildfire in western Canada, we initiated a multi-faceted water quality monitoring program that suggested brief (hours to days) wildfire signatures could be detected in several large river systems, particularly following rainfall events greater than 10 mm. Continuous monitoring of flow and water quality showed distinct, precipitation-associated signatures of ash transport in rivers draining expansive (800-100,000 km2) and partially-burned (<1-22 percent burned) watersheds, which were not evident in nearby unburned regions. Yields of suspended sediment, nutrients (nitrogen, phosphorus) and metals (lead, others) from impacted rivers were 1.2-10 times greater than from those draining unburned regions. Post-fire suspended sediment concentrations in impacted rivers were often larger than pre-fire 95% prediction intervals based on several years of water sampling. These multiple lines of evidence indicate that low-relief landscapes can mobilize wildfire-related material to rivers similarly, though less-intensively and over shorter durations, than headwater regions. We propose that uneven mixing of heavily-impacted tributaries with high-order rivers may partially explain detection of wildfire signals in these large systems that may impact downstream water users.

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.

Impact of visual callosal pathway is dependent upon ipsilateral thalamus.

The visual callosal pathway, which reciprocally connects the primary visual cortices, is thought to play a pivotal role in cortical binocular processing. In rodents, the functional role of this pathway is largely unknown. Here, we measure visual cortex spiking responses to visual stimulation using population calcium imaging and functionally isolate visual pathways originating from either eye. We show that callosal pathway inhibition significantly reduced spiking responses in binocular and monocular neurons and abolished spiking in many cases. However, once isolated by blocking ipsilateral visual thalamus, callosal pathway activation alone is not sufficient to drive evoked cortical responses. We show that the visual callosal pathway relays activity from both eyes via both ipsilateral and contralateral visual pathways to monocular and binocular neurons and works in concert with ipsilateral thalamus in generating stimulus evoked activity. This shows a much greater role of the rodent callosal pathway in cortical processing than previously thought.

Erosion of the Alberta badlands produces highly variable and elevated heavy metal concentrations in the Red Deer River, Alberta.

Erosion is important in the transport of heavy metals from terrestrial to fluvial environments. In this study, we investigated riverine heavy metal (Cd, Cu, Hg and Pb) dynamics in the Red Deer River (RDR) watershed at sites upstream (n=2) and downstream (n=7) of the Alberta badlands, an area of naturally high erosion. At sites draining the badlands, total water column Cd, Cu, Hg and Pb concentrations frequently exceeded guidelines for the protection of freshwater biota. Furthermore, peak concentrations of total Cd (9.8µgL-1), Cu (212µgL-1), Hg (649ngL-1) and Pb (361µgL-1) were higher than, or comparable to, values reported for rivers and streams heavily impacted by anthropogenic activities. Total suspended solids (TSS) explained a large proportion (r2=0.34-0.83) of the variation in total metal concentrations in the RDR and tributaries and metal fluxes were dominated by the particulate fraction (60-98%). Suspended sediment concentrations (Csed) and metal to aluminum ratios were generally not indicative of substantial sediment enrichment. Rather, the highly variable and elevated metal concentrations in the RDR watershed were a function of the high and variable suspended sediment fluxes which characterize the river system. While the impact of this on aquatic biota requires further investigation, we suggest erosion in the Alberta badlands may be contributing to Hg-based fish consumption advisories in the RDR. Importantly, this highlights a broader need for information on contaminant dynamics in watersheds subject to elevated rates of erosion.

Behavioral Neuroscience: Who's Afraid of the C57BL/6 Mouse?

Behavioral paradigms in which laboratory rodents express behaviors that their wild counterparts presumably need every day are rare: a novel prey-capture model for laboratory mice has been developed for examining the neurophysiological underpinnings of prey capture in mice.

Primate Thalamus: More Than Meets an Eye.

A recent study shows conclusively that the koniocellular layers of the marmoset dorsal lateral geniculate nucleus have binocularly responsive neurons. This adds a new twist to the traditional view about binocular processing in the primate visual system and raises questions about the role of dorsal lateral geniculate nucleus in early binocular processing.

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.

Flavonoid metabolites reduce tumor necrosis factor-α secretion to a greater extent than their precursor compounds in human THP-1 monocytes.

SCOPE: Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti-inflammatory effects of flavonoid metabolites relative to their precursor structures. METHODS AND RESULTS: Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1-10 µM) were screened for their ability to reduce LPS-induced tumor necrosis factor-α (TNF-α) secretion in THP-1 monocytes. One micromolar peonidin-3-glucoside, cyanidin-3-glucoside, and the metabolites isovanillic acid (IVA), IVA-glucuronide, vanillic acid-glucuronide, protocatechuic acid-3-sulfate, and benzoic acid-sulfate significantly reduced TNF-α secretion when in isolation, while there was no effect on TNF-α mRNA expression. Four combinations of metabolites that included 4-hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF-α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS-induced IL-1ß and IL-10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL-1ß secretion but none of the flavonoids or metabolites significantly modified IL-10 secretion. CONCLUSION: This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites.

Imaging neuronal population activity in awake and anesthetized rodents.

Recent advances in in vivo two-photon imaging have extended the technique to permit the detection of action potentials (APs) in populations of spatially resolved neurons in awake animals. Although experimentally demanding, this technique's potential applications include experiments to investigate perception, behavior, and other awake states. Here we outline experimental procedures for imaging neuronal populations in awake and anesthetized rodents. Details are provided on habituation to head fixation, surgery, head plate design, and dye injection. Determination of AP detection accuracy through simultaneous optical and electrophysiological recordings is also discussed. Basic problems of data analysis are considered, such as correction of signal background and baseline drift, AP detection, and motion correction. As an application of the method, the comparison of neuronal activity across arousal states is considered in detail, and some future directions are discussed.