Open-source statistical and data processing tools for wide-field optical imaging data in mice

SIGNIFICANCE: Wide-field optical imaging (WOI) can produce concurrent hemodynamic and cell-specific calcium recordings across the entire cerebral cortex in animal models. There have been multiple studies using WOI to image mouse models with various environmental or genetic manipulations to understand various diseases. Despite the utility of pursuing mouse WOI alongside human functional magnetic resonance imaging (fMRI), and the multitude of analysis toolboxes in the fMRI literature, there is not an available open-source, user-friendly data processing and statistical analysis toolbox for WOI data. AIM: To assemble a MATLAB toolbox for processing WOI data, as described and adapted to combine techniques from multiple WOI groups and fMRI. APPROACH: We outline our MATLAB toolbox on GitHub with multiple data analysis packages and translate a commonly used statistical approach from the fMRI literature to the WOI data. To illustrate the utility of our MATLAB toolbox, we demonstrate the ability of the processing and analysis framework to detect a well-established deficit in a mouse model of stroke and plot activation areas during an electrical paw stimulus experiment. RESULTS: Our processing toolbox and statistical methods isolate a somatosensory-based deficit 3 days following photothrombotic stroke and cleanly localize sensory stimulus activations. CONCLUSIONS: The toolbox presented here details an open-source, user-friendly compilation of WOI processing tools with statistical methods to apply to any biological question investigated with WOI techniques

Wide-field optical imaging of neurological disorders and sleep in mice

Neuroimaging has revolutionized the way in which we understand the hierarchical organization of the amazingly complex, interconnected human brain. Neuroimaging techniques, like functional magnetic resonance imaging (fMRI), have provided high quality structural and functional data, providing multiple in-depth analyses and biomarkers of disease processes. In animal models, mechanistic studies can uncover root pathologies that aren’t explorable in humans. In mice, brain functional connectivity (FC) can be measured via Optical Intrinsic Signal (OIS) imaging – a modality that measures vascular reactivity as a surrogate for neural activity via quantification of fluctuations in oxygenated-hemoglobin (similar to the blood oxygen level dependent (BOLD) signal used in fMRI). Another advantage of optical neuroimaging in mice is the expression of genetically encoded calcium indicators (GECIs), which provide cell-specific and network-level functional imaging of brain activity at speeds up to at least 4Hz. Imaging in higher frequency bands (compared to \u3c0.2Hz in fMRI or other hemoglobin-based imaging modalities) allows for resolution of neural specific phenomena on the order of milliseconds, such as the global ∼1Hz slow oscillation that is characteristic of anesthesia and non-rapid eye movement (NREM) sleep. We imaged mice expressing the GECI GCaMP6 in excitatory neurons while awake, in NREM (verified by EEG), or under ketamine/xylazine (K/X) or Dexmedetomidine (Dex) anesthesia and reconcile discrepancies between activity dynamics observed with hemoglobin vs. calcium (GCaMP6) imaging. Alterations in correlation structure were most obvious in delta band calcium NREM and anesthesia data, resulting in maps with large regions of polarized positive and negative correlations covering the field-of-view (FOV). We use principal component analysis (PCA) to provide evidence that the slow oscillation superimposes on FC rather than replaces FC patterns typical of the alert state. While consciousness state can oscillate on the order of seconds, many studies of disease processes are most informative across a longer period of time. Surgical preparations coupled with optical imaging allow for longitudinal experiments on varying timescales. For example, sequalae of subarachnoid hemorrhage (SAH) include vasospasm, microvessel thrombi, and other delayed cerebral ischemic (DCI) events around 3 days post SAH. These DCI events have been shown to coincide with up-regulation of the neuroprotective peptide Sirtuin1 (SIRT1), using an endovascular perforation mouse model. Here, we display global FC disruption caused by SAH and DCI events in parallel with behavioral deterioration. Normal brain connectivity and behavior was maintained during SAH and DCI via two different treatments targeting SIRT1 activation. SIRT1-specific (resveratrol) and non-specific (hypoxic conditioning) treatments both protected against the FC deficits induced by SAH and DCI, with the latter providing the largest protective effect. This indicates that conditioning-based strategies targeting SIRT1-directed mechanisms provide multifaceted neurovascular protection in experimental SAH – data that further supports the overarching hypothesis that conditioning- based therapy is a powerful approach with great potential for improving patient outcome after aneurysmal SAH. Studies involving focal injury (e.g., stroke, SAH) usually exhibit functional deficits surrounding the injured tissue, however, it is less clear how diffuse processes, such as novel models of acute septic encephalopathy (i.e., Delirium), and encephalitis caused by Zika virus infection, alter brain dynamics. Septic encephalopathy leads to major and costly burdens for a large percentage of admitted hospital patients. Elderly patients are at an increased risk, especially those with dementia. Current treatments are aimed at sedation to combat mental status changes and are not aimed at the underlying cause of encephalopathy. Indeed, the underlying pathology linking together peripheral infection and altered neural function has not been established, largely because good, acutely accessible readouts of encephalopathy in animal models do not exist. In-depth behavioral testing in animals lasts multiple days, outlasting the time frame of acute encephalopathy. Here, we propose optical fluorescent imaging of neural FC as a readout of encephalopathy in a mouse model of acute sepsis. Imaging and basic behavioral assessment was performed at baseline, Hr8, Hr24, and Hr72 following injection of either lipopolysaccharide (LPS) or phosphate buffered saline (PBS). Neural FC strength decreased at Hr8 and returned to baseline by Hr72 in somatosensory and parietal cortical regions. Additionally, neural fluctuations transiently declined at Hr8 and returned to baseline by Hr72. Both FC strength and neural fluctuation tone correlated with behavioral neuroscore indicating this imaging methodology is a sensitive and acute readout of encephalopathy. Zika virus (ZIKV) emerged as a prominent global health concern due to the severe neurologic injury in infants born to adults who had ZIKV infection during pregnancy. However, neurologic manifestations in healthy adults were subsequently reported during Zika pandemics in South America and Southeast Asia. In this population, infection can result in severe cases of encephalitis and have lasting impacts on cognition, and learning and memory, even after recovery from acute infection. Recent studies have uncovered extensive ZIKV- related neural apoptosis within the trisynaptic circuit involving the entorhinal cortex, the cornu ammonis, and the dentate gyrus of the hippocampus in adult mice. However, there are many contributing regions and circuits involved in cognition and learning and memory outside of this trisynaptic circuit. Communication within the cortex and between the cortex and hippocampus is necessary for a variety of neurological processes, such as performing cognitive tasks or for memory consolidation during sleep. Here, we investigate cortical networks and connectivity utilizing wide-field optical fluorescence imaging. We demonstrate that functional deficits congregate in regions of cortex that are highly communicative with hippocampus, such as somatosensory and retrosplenial cortices. Further, we prove that these functional imaging deficits are correlated with other metrics of disease severity, such as encephalitis score and increased delta power, providing a potentially useful clinical biomarker of disease. Finally, these imaging deficits resolve after recovery from acute infection. While optical methods have obvious advantages when used to study animal models, the technique is relatively novel (compared to fMRI) therefore, there are many avenues for data processing algorithms to improve. Similar to fMRI, historically, optical methods use a remarkably simple bivariate Pearson-based approach to mapping FC, leading to quick and easy-to-interpret models of brain networks but also susceptibility to global sources of variance (e.g., motion, Mayer waves). Previously, we demonstrated the binarizing effect of the slow oscillation on FC during NREM and K/X anesthesia. While PCA effectively removed the slow oscillation, it is reasonable to assume that a biological process cannot be completely explained in algebraically orthogonal components. Therefore, we pioneer a multivariate approach to imputing individual neural networks from spontaneous neuroimaging data in mice in an effort to map connectivity with less susceptibility to confounding variance. Calcium dynamics in all brain pixels are holistically weighted via support vector regression to predict activity in a region of interest (ROI). This approach yielded remarkably high prediction accuracy, suggesting the optimized pixel weights represent multivariate functional connectivity (MFC) strength with the ROI. Additionally, MFC maps were largely impervious to the slow oscillation. Moreover, MFC maps more closely aligned with anatomical connectivity as modeled through axonal projection images, than FC maps. Lastly, MFC analysis provided a more powerful connectivity deficit detection following stroke compared to standard FC. These results show that MFC has several performance and conceptual advantages over standard FC and should be considered more broadly within the FC analysis community. Further, with study of diffuse processes (e.g., LPS and ZIKV infection), statistical developments are crucial to solve the multiple comparisons problem when examining all cortical regions within the FOV. Therefore, part of this thesis focuses on the development of a streamlined, open source, user friendly data processing toolbox that contains multiple statistical approaches to make the aforementioned studies possible. Together, the following presents the multiple ways wide-field optical imaging can be used to learn more about the brain’s functional architecture in health and disease

Maternal fluoxetine exposure alters cortical hemodynamic and calcium response of offspring to somatosensory stimuli

Epidemiological studies have found an increased incidence of neurodevelopmental disorders in populations prenatally exposed to selective serotonin reuptake inhibitors (SSRIs). Optical imaging provides a minimally invasive way to determine if perinatal SSRI exposure has long-term effects on cortical function. Herein we probed the functional neuroimaging effects of perinatal SSRI exposure in a fluoxetine (FLX)-exposed mouse model. While resting-state homotopic contralateral functional connectivity was unperturbed, the evoked cortical response to forepaw stimulation was altered in FLX mice. The stimulated cortex showed decreased activity for FLX versus controls, by both hemodynamic responses [oxyhemoglobin (Hb

Cortex-wide, cellular-resolution two-photon microscopy

Functional imaging of the mouse brain in its extreme complexity involves substantial trade-offs. An optical intrinsic spectroscopy system can image the entire cortex but at the expense of spatial and temporal resolution [1]. A two-photon microscope (TPM) can image single neurons with high temporal resolution, but the field of view (FOV) is generally restricted. Advanced techniques like random-access scanning allow for imaging single neurons that are millimeters apart but only by ignoring the neurons and tissue in between [2]. By carefully considering the properties of the optical components as well as the imaging requirements, we present a TPM capable of imaging nearly the entire mouse cortex with 15 Hz frame rates and single neuron resolution. Please click Additional Files below to see the full abstract

Automated sleep state classification of wide-field calcium imaging data via multiplex visibility graphs and deep learning

BACKGROUND: Wide-field calcium imaging (WFCI) allows for monitoring of cortex-wide neural dynamics in mice. When applied to the study of sleep, WFCI data are manually scored into the sleep states of wakefulness, non-REM (NREM) and REM by use of adjunct EEG and EMG recordings. However, this process is time-consuming and often suffers from low inter- and intra-rater reliability and invasiveness. Therefore, an automated sleep state classification method that operates on WFCI data alone is needed. NEW METHOD: A hybrid, two-step method is proposed. In the first step, spatial-temporal WFCI data is mapped to multiplex visibility graphs (MVGs). Subsequently, a two-dimensional convolutional neural network (2D CNN) is employed on the MVGs to be classified as wakefulness, NREM and REM. RESULTS: Sleep states were classified with an accuracy of 84% and Cohen\u27s κ of 0.67. The method was also effectively applied on a binary classification of wakefulness/sleep (accuracy=0.82, κ = 0.62) and a four-class wakefulness/sleep/anesthesia/movement classification (accuracy=0.74, κ = 0.66). Gradient-weighted class activation maps revealed that the CNN focused on short- and long-term temporal connections of MVGs in a sleep state-specific manner. Sleep state classification performance when using individual brain regions was highest for the posterior area of the cortex and when cortex-wide activity was considered. COMPARISON WITH EXISTING METHOD: On a 3-hour WFCI recording, the MVG-CNN achieved a κ of 0.65, comparable to a κ of 0.60 corresponding to the human EEG/EMG-based scoring. CONCLUSIONS: The hybrid MVG-CNN method accurately classifies sleep states from WFCI data and will enable future sleep-focused studies with WFCI

Endothelial ether lipids link the vasculature to blood pressure, behavior, and neurodegeneration

Vascular disease contributes to neurodegeneration, which is associated with decreased blood pressure in older humans. Plasmalogens, ether phospholipids produced by peroxisomes, are decreased in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. However, the mechanistic links between ether phospholipids, blood pressure, and neurodegeneration are not fully understood. Here, we show that endothelium-derived ether phospholipids affect blood pressure, behavior, and neurodegeneration in mice. In young adult mice, inducible endothelial-specific disruption of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, unexpectedly decreased circulating plasmalogens. PexRAP endothelial knockout (PEKO) mice responded normally to hindlimb ischemia but had lower blood pressure and increased plasma renin activity. In PEKO as compared with control mice, tyrosine hydroxylase was decreased in the locus coeruleus, which maintains blood pressure and arousal. PEKO mice moved less, slept more, and had impaired attention to and recall of environmental events as well as mild spatial memory deficits. In PEKO hippocampus, gliosis was increased, and a plasmalogen associated with memory was decreased. Despite lower blood pressure, PEKO mice had generally normal homotopic functional connectivity by optical neuroimaging of the cerebral cortex. Decreased glycogen synthase kinase-3 phosphorylation, a marker of neurodegeneration, was detected in PEKO cerebral cortex. In a co-culture system, PexRAP knockdown in brain endothelial cells decreased glycogen synthase kinase-3 phosphorylation in co-cultured astrocytes that was rescued by incubation with the ether lipid alkylglycerol. Taken together, our findings suggest that endothelium-derived ether lipids mediate several biological processes and may also confer neuroprotection in mice

Biophysical Characteristics of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Structure and Chemistry Lead to Differential Toxicity

The biophysical chemistry of macromolecular complexes confer their functional characteristics. We investigate the mechanisms that make the AB5 holotoxin of Vibrio cholerae (CT) a significantly more pathogenic molecule than the enterotoxin of Escherichia coli (LT) with which it shares 88% similarity and whose structure is homologous with a backbone RMSD of 0.84 Å and imposes its deleterious effects though the same process to constitutively ADP-ribosylate adenylate cyclase. We present computational data that characterizes the impact of amino acid variations in the A2 tail, which helps to explain experimental data that demonstrate CT’s higher toxicity. A hydrophobic patch on the B pentamer interface and its interactions with the A subdomain are partially disrupted by the substitution of an aspartic acid (LT) for glycine in CT. CT’s holotoxin has less solvent accessible surface area (94 Ų vs 54 Ų) and higher contact area (280 Ų vs 241 Ų) with S228, which is a gatekeeper, partially controlling the diffusion of water into the pore. CT excludes water from the top of the central pore whereas LT allows much more water to interact. These biophysical properties of the toxins lead to their differential toxicity and resulting impact to human health

Functional connectivity structure of cortical calcium dynamics in anesthetized and awake mice.

The interplay between hemodynamic-based markers of cortical activity (e.g. fMRI and optical intrinsic signal imaging), which are an indirect and relatively slow report of neural activity, and underlying synaptic electrical and metabolic activity through neurovascular coupling is a topic of ongoing research and debate. As application of resting state functional connectivity measures is extended further into topics such as brain development, aging and disease, the importance of understanding the fundamental physiological basis for functional connectivity will grow. Here we extend functional connectivity analysis from hemodynamic- to calcium-based imaging. Transgenic mice (n = 7) expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketamine/xylazine) and awake states. Sequential LED illumination (λ = 454, 523, 595, 640nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. Functional connectivity network maps were constructed for infraslow (0.009-0.08Hz), intermediate (0.08-0.4Hz), and high (0.4-4.0Hz) frequency bands. At infraslow and intermediate frequencies, commonly used in BOLD fMRI and fcOIS studies of functional connectivity and implicated in neurovascular coupling mechanisms, GCaMP6 and HbO2 functional connectivity structures were in high agreement, both qualitatively and also quantitatively through a measure of spatial similarity. The spontaneous dynamics of both contrasts had the highest correlation when the GCaMP6 signal was delayed with a ~0.6-1.5s temporal offset. Within the higher-frequency delta band, sensitive to slow wave sleep oscillations in non-REM sleep and anesthesia, we evaluate the speed with which the connectivity analysis stabilized and found that the functional connectivity maps captured putative network structure within time window lengths as short as 30 seconds. Homotopic GCaMP6 functional connectivity maps at 0.4-4.0Hz in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. This structure is potentially explained in part by observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas. During awake imaging, this spatio-temporal quality is altered, and a more complex and detailed functional connectivity structure is observed. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and neurovascular coupling and provide a framework for subsequent studies of neurological disease such as stroke