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

In utero exposure to transient ischemia-hypoxemia promotes long-term neurodevelopmental abnormalities in male rat offspring

The impact of transient ischemic-hypoxemic insults on the developing fetal brain is poorly understood despite evidence suggesting an association with neurodevelopmental disorders such as schizophrenia and autism. To address this, we designed an aberrant uterine hypercontractility paradigm with oxytocin to better assess the consequences of acute, but transient, placental ischemia-hypoxemia in term pregnant rats. Using MRI, we confirmed that oxytocin-induced aberrant uterine hypercontractility substantially compromised uteroplacental perfusion. This was supported by the observation of oxidative stress and increased lactate concentration in the fetal brain. Genes related to oxidative stress pathways were significantly upregulated in male, but not female, offspring 1 hour after oxytocin-induced placental ischemia-hypoxemia. Persistent upregulation of select mitochondrial electron transport chain complex proteins in the anterior cingulate cortex of adolescent male offspring suggested that this sex-specific effect was enduring. Functionally, offspring exposed to oxytocin-induced uterine hypercontractility showed male-specific abnormalities in social behavior with associated region-specific changes in gene expression and functional cortical connectivity. Our findings, therefore, indicate that even transient but severe placental ischemia-hypoxemia could be detrimental to the developing brain and point to a possible mitochondrial link between intrauterine asphyxia and neurodevelopmental disorders

Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke

Understanding circuit-level manipulations that affect the brain\u27s capacity for plasticity will inform the design of targeted interventions that enhance recovery after stroke. Following stroke, increased contralesional activity (e.g. use of the unaffected limb) can negatively influence recovery, but it is unknown which specific neural connections exert this influence, and to what extent increased contralesional activity affects systems- and molecular-level biomarkers of recovery. Here, we combine optogenetic photostimulation with optical intrinsic signal imaging to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice either recovered spontaneously or received chronic optogenetic excitation of right S1FP over the course of 4 weeks. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of stimulus-evoked activity in the unaffected limb. This maneuver also prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited transcriptional changes in several genes relevant for recovery. Our results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of cortical activity and several neuroplasticity-related genes after stroke, and highlight the importance of site selection for targeted therapeutic interventions after focal ischemia

Effective connectivity measured using optogenetically evoked hemodynamic signals exhibits topography distinct from resting state functional connectivity in the mouse

Brain connectomics has expanded from histological assessment of axonal projection connectivity (APC) to encompass resting state functional connectivity (RS-FC). RS-FC analyses are efficient for whole-brain mapping, but attempts to explain aspects of RS-FC (e.g., interhemispheric RS-FC) based on APC have been only partially successful. Neuroimaging with hemoglobin alone lacks specificity for determining how activity in a population of cells contributes to RS-FC. Wide-field mapping of optogenetically defined connectivity could provide insights into the brain\u27s structure-function relationship. We combined optogenetics with optical intrinsic signal imaging to create an efficient, optogenetic effective connectivity (Opto-EC) mapping assay. We examined EC patterns of excitatory neurons in awake, Thy1-ChR2 transgenic mice. These Thy1-based EC (Thy1-EC) patterns were evaluated against RS-FC over the cortex. Compared to RS-FC, Thy1-EC exhibited increased spatial specificity, reduced interhemispheric connectivity in regions with strong RS-FC, and appreciable connection strength asymmetry. Comparing the topography of Thy1-EC and RS-FC patterns to maps of APC revealed that Thy1-EC more closely resembled APC than did RS-FC. The more general method of Opto-EC mapping with hemoglobin can be determined for 100 sites in single animals in under an hour, and is amenable to other neuroimaging modalities. Opto-EC mapping represents a powerful strategy for examining evolving connectivity-related circuit plasticity

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

Evaluation of gliovascular functions of AQP4 readthrough isoforms

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels

Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke

Understanding circuit-level manipulations that affect the brain’s capacity for plasticity will inform the design of targeted interventions that enhance recovery after stroke. Following stroke, increased contralesional activity (e.g. use of the unaffected limb) can negatively influence recovery, but it is unknown which specific neural connections exert this influence, and to what extent increased contralesional activity affects systems-and molecular-level biomarkers of recovery. Here, we combine optogenetic photostimulation with optical intrinsic signal imaging (OISI) to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice either recovered spontaneously or received chronic optogenetic excitation of right S1FP over the course of 4 weeks. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of stimulus-evoked activity in the unaffected limb. This maneuver also prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally-distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited transcriptional changes in several genes relevant for recovery. Our results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of cortical activity and several neuroplasticity-related genes after stroke, and highlight the importance of site selection for therapeutic intervention after focal ischemia

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