Cellular layering is a hallmark of the mammalian neocortex. A key challenge in studying circuit function within the neocortex is to understand the spatial and temporal patterns of information flow between different columns and layers. In the paper, a mesoscopic functional imaging method for neural activities in mouse cortex in vivo was developed and demonstrated. We demonstrated the depth-resolved capability of 3D mesoscopic imaging by photostimulating the layer 5 neurons in Thy1-ChR2-YFP transgenic mice and further recorded the layer-specific functional projections between primary somatosensory cortex and primary motor cortex in vivo, following single whisker deflection.
KEYWORDS: Neurons, Photostimulation, Luminescence, In vivo imaging, Brain, 3D image processing, Sensors, Neuroimaging, Voltage sensitive dyes, Imaging systems
Significance: Cellular layering is a hallmark of the mammalian neocortex with layer and cell type-specific connections within the cortical mantle and subcortical connections. A key challenge in studying circuit function within the neocortex is to understand the spatial and temporal patterns of information flow between different columns and layers.
Aim: We aimed to investigate the three-dimensional (3D) layer- and area-specific interactions in mouse cortex in vivo.
Approach: We applied a new promising neuroimaging method—fluorescence laminar optical tomography in combination with voltage-sensitive dye imaging (VSDi). VSDi is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but it is traditionally used for two-dimensional (2D) imaging. Our mesoscopic technique allows visualization of neuronal activity in a 3D manner with high temporal resolution.
Results: We first demonstrated the depth-resolved capability of 3D mesoscopic imaging technology in Thy1-ChR2-YFP transgenic mice. Next, we recorded the long-range functional projections between sensory cortex (S1) and motor cortex (M1) in mice, in vivo, following single whisker deflection.
Conclusions: The results show that mesoscopic imaging technique has the potential to investigate the layer-specific neural connectivity in the mouse cortex in vivo. Combination of mesoscopic imaging technique with optogenetic control strategy is a promising platform for determining depth-resolved interactions between cortical circuit elements.
Understanding the functional wiring of neural circuits and their patterns of activation following sensory stimulations is a fundamental task in the field of neuroscience. Furthermore, charting the activity patterns is undoubtedly important to elucidate how neural networks operate in the living brain. However, optical imaging must overcome the effects of light scattering in the tissue, which limit the light penetration depth and affect both the imaging quantitation and sensitivity. Laminar optical tomography (LOT) is a three-dimensional (3-D) in-vivo optical imaging technique that can be used for functional imaging. LOT can achieve both a resolution of 100 to 200 μm and a penetration depth of 2 to 3 mm based either on absorption or fluorescence contrast, as well as large field-of-view and high acquisition speed. These advantages make LOT suitable for 3-D depth-resolved functional imaging of the neural functions in the brain and spinal cords. We review the basic principles and instrumentations of representative LOT systems, followed by recent applications of LOT on 3-D imaging of neural activities in the rat forepaw stimulation model and mouse whisker-barrel system.
Visualization of whole brain activity during epileptic seizures is essential for both fundamental research into the disease mechanisms and the development of efficient treatment strategies. It has been previously discussed that pathological synchronization originating from cortical areas may reinforce backpropagating signaling from the thalamic neurons, leading to massive seizures through enhancement of high frequency neural activity in the thalamocortical loop. However, the study of deep brain neural activity is challenging with the existing functional neuroimaging methods due to lack of adequate spatiotemporal resolution or otherwise insufficient penetration into subcortical areas. To investigate the role of thalamocortical activity during epileptic seizures, we developed a new functional neuroimaging framework based on spatiotemporal correlation of volumetric optoacoustic hemodynamic responses with the concurrent electroencephalogram recordings and anatomical brain landmarks. The method is shown to be capable of accurate three-dimensional mapping of the onset, spread, and termination of the epileptiform events in a 4-aminopyridine acute model of focal epilepsy. Our study is the first to demonstrate entirely noninvasive real-time visualization of synchronized epileptic foci in the whole mouse brain, including the neocortex and subcortical structures, thus opening new vistas in systematic studies toward the understanding of brain signaling and the origins of neurological disorders.
To control the overall action of the body, brain consumes a large amount of energy in proportion to its volume. In
humans and many other species, the brain gets most of its energy from oxygen-dependent metabolism of glucose. An
abnormal metabolic rate of glucose and/or oxygen usually reflects a diseased status of brain, such as cancer or
Alzheimer’s disease. We have demonstrated the feasibility of imaging mouse brain metabolism using photoacoustic
computed tomography (PACT), a fast, noninvasive and functional imaging modality with optical contrast and acoustic
resolution. Brain responses to forepaw stimulations were imaged transdermally and transcranially. 2-NBDG, which
diffuses well across the blood-brain-barrier, provided exogenous contrast for photoacoustic imaging of glucose response.
Concurrently, hemoglobin provided endogenous contrast for photoacoustic imaging of hemodynamic response. Glucose
and hemodynamic responses were quantitatively unmixed by using two-wavelength measurements. We found that
glucose uptake and blood perfusion around the somatosensory region of the contralateral hemisphere were both
increased by stimulations, indicating elevated neuron activity. The glucose response amplitude was about half that of the
hemodynamic response. While the glucose response area was more homogenous and confined within the somatosensory
region, the hemodynamic response area showed a clear vascular pattern and spread about twice as wide as that of the
glucose response. The PACT of mouse brain metabolism was validated by high-resolution open-scalp OR-PAM and
fluorescence imaging. Our results demonstrate that 2-NBDG-enhanced PACT is a promising tool for noninvasive studies
of brain metabolism.
Advances in the functional imaging of cortical hemodynamics have greatly facilitated the understanding of neurovascular coupling. In this study, label-free optical-resolution photoacoustic microscopy (OR-PAM) was used to monitor microvascular responses to direct electrical stimulations of the mouse somatosensory cortex through a cranial opening. The responses appeared in two forms: vasoconstriction and vasodilatation. The transition between these two forms of response was observed in single vessels by varying the stimulation intensity. Marked correlation was found between the current-dependent responses of two daughter vessels bifurcating from the same parent vessel. Statistical analysis of twenty-seven vessels from three different animals further characterized the spatial-temporal features and the current dependence of the microvascular response. Our results demonstrate that OR-PAM is a valuable tool to study neurovascular coupling at the microscopic level.
Advances in the brain functional imaging greatly facilitated the understanding of neurovascular coupling. For monitoring
of the microvascular response to the brain electrical stimulation in vivo we used optical-resolution photoacoustic
microscopy (OR-PAM) through the cranial openings as well as transcranially. Both types of the vascular response,
vasoconstriction and vasodilatation, were clearly observed with good spatial and temporal resolution. Obtained results
confirm one of the primary points of the neurovascular coupling theory that blood vessels could present vasoconstriction
or vasodilatation in response to electrical stimulation, depending on the balance between inhibition and excitation of the
different parts of the elements of the neurovascular coupling system.
Optical-resolution photoacoustic microscopy (OR-PAM) is applied to functional brain imaging in living mice. A near-diffraction-limited bright-field optical illumination is employed to achieve micrometer lateral resolution, and a dual-wavelength measurement is utilized to extract the blood oxygenation information. The variation in hemoglobin oxygen saturation (sO2) along vascular branching has been imaged in a precapillary arteriolar tree and a postcapillary venular tree, respectively. To the best of our knowledge, this is the first report on in vivo volumetric imaging of brain microvascular morphology and oxygenation down to single capillaries through intact mouse skulls. It is anticipated that: (i) chronic imaging enabled by this minimally invasive procedure will advance the study of cortical plasticity and neurological diseases; (ii) revealing the neuroactivity-dependent changes in hemoglobin concentration and oxygenation will facilitate the understanding of neurovascular coupling at the capillary level; and (iii) combining functional OR-PAM and high-resolution blood flowmetry will have the potential to explore cellular pathways of brain energy metabolism.
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