Ultra-deep through-skull mouse brain imaging via the combination of skull optical clearing and three-photon microscopy

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and thee-photon fluorescence microscopy (3PM). The Visible-NIR-II compatible Skull Optical Clearing Agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1 mm and neuronal imaging depth of >700 μm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

Awake mouse brain photoacoustic and optical imaging through a transparent ultrasound cranial window

Optical resolution photoacoustic microscopy (OR-PAM) can map the cerebral vasculature at capillary level resolution. However, the OR-PAM setup’s bulky imaging head makes awake mouse brain imaging challenging and inhibits its integration with other optical neuroimaging modalities. Moreover, the glass cranial windows used for optical microscopy are unsuitable for OR-PAM due to the acoustic impedance mismatch between the glass plate and the tissue. To overcome these challenges, we propose a lithium niobate based transparent ultrasound trans-ducer (TUT) as a cranial window on a thinned mouse skull. The TUT cranial window simplifies the imaging head considerably due to its dual functionality as an optical window and ultrasound transducer. The window remains stable for six weeks, with no noticeable inflammation and minimal bone regrowth. The TUT window’s potential is demonstrated by imaging the awake mouse cerebral vasculature using OR-PAM, intrinsic optical signal imaging and two-photon microscopy. The TUT cranial window can potentially also be used for ultrasound stimulation and simultaneous multimodal imaging of the awake mouse brain.

Miniscope-LFOV: A large field of view, single cell resolution, miniature microscope for wired and wire-free imaging of neural dynamics in freely behaving animals

We present a large field of view (FOV) open-source miniature microscope (MiniLFOV) designed to extend the capabilities of the UCLA Miniscope platform to large-scale, single cell resolution neural imaging in freely behaving large rodents and head-fixed mice. This system is capable of multiple imaging configurations, including deep brain imaging using implanted optical probes and cortical imaging through cranial windows. The MiniLFOV interfaces with existing open-source UCLA Miniscope DAQ hardware and software, can achieve single cell resolution imaging across a 3.6 × 2.7 mm field of view at 23 frames per second, has an electrically adjustable working distance of up to 3.5 mm±150 µm using an onboard electrowetting lens, incorporates an absolute head-orientation sensor, and weighs under 14 grams. The MiniLFOV provides a 30-fold larger FOV and yields 20-fold better sensitivity than Miniscope V3, and a 12-fold larger FOV with 2-fold better sensitivity than Miniscope V4. Power and data transmission are handled through a single, flexible coaxial cable down to 0.3 mm in diameter facilitating naturalistic behavior. We validated the MiniLFOV in freely behaving rats by simultaneously imaging >1000 GCaMP7s expressing neurons in the CA1 layer of the hippocampus and in head-fixed mice by simultaneously imaging ~2000 neurons in the mouse dorsal cortex through a 4 × 4 mm cranial window. For freely behaving experiments, the MiniLFOV supports optional wire-free operation using a 3.5 g wire-free data acquisition expansion board which enables close to 1-hour of wire-free recording with a 400 mAh (7.5 g) on-board single-cell lithium-polymer battery and expands wire-free imaging techniques to larger animal models. We expect this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

Simultaneous Two-Photon Voltage or Calcium Imaging and Multi-Channel Local Field Potential Recordings in Barrel Cortex of Awake and Anesthetized Mice

Neuronal population activity, both spontaneous and sensory-evoked, generates propagating waves in cortex. However, high spatiotemporal-resolution mapping of these waves is difficult as calcium imaging, the work horse of current imaging, does not reveal subthreshold activity. Here, we present a platform combining voltage or calcium two-photon imaging with multi-channel local field potential (LFP) recordings in different layers of the barrel cortex from anesthetized and awake head-restrained mice. A chronic cranial window with access port allows injecting a viral vector expressing GCaMP6f or the voltage-sensitive dye (VSD) ANNINE-6plus, as well as entering the brain with a multi-channel neural probe. We present both average spontaneous activity and average evoked signals in response to multi-whisker air-puff stimulations. Time domain analysis shows the dependence of the evoked responses on the cortical layer and on the state of the animal, here separated into anesthetized, awake but resting, and running. The simultaneous data acquisition allows to compare the average membrane depolarization measured with ANNINE-6plus with the amplitude and shape of the LFP recordings. The calcium imaging data connects these data sets to the large existing database of this important second messenger. Interestingly, in the calcium imaging data, we found a few cells which showed a decrease in calcium concentration in response to vibrissa stimulation in awake mice. This system offers a multimodal technique to study the spatiotemporal dynamics of neuronal signals through a 3D architecture in vivo. It will provide novel insights on sensory coding, closing the gap between electrical and optical recordings.

EXTH-26. LAYER-BY-LAYER NANOPARTICLES DESIGNED FOR DUAL BLOOD-BRAIN BARRIER AND GLIOMA TARGETING

BACKGROUND While biologically diverse, high grade gliomas (HGGs) have a dismal prognosis in both adults and children. Promising therapeutics have been identified for HGGs based on common genomic alterations and aberrant signaling pathways, but achieving effective drug exposure at the tumor site remains a challenge largely due to the blood-brain barrier (BBB). HYPOTHESIS: A tunable nanocarrier platform can improve nanoparticle delivery across the (BBB) and into glioma cells. METHODS We synthesized layer-by-layer nanoparticles by coating anionic, fluorescent liposomes with nanometers-thick layers of oppositely charged polyelectrolytes, creating a library of organic, nontoxic drug carriers with varied surface chemistries. We characterized the library using dynamic light scattering and quantified interactions with a range of pediatric and adult glioma cell lines using flow cytometry. We used intravital two-photon microscopy to quantify nanoparticle trafficking across the intact BBB through a cranial window in anesthetized mice. RESULTS Nanoparticle surface chemistry strongly influences cellular trafficking in vitro, with two polymers identified as particularly high-performing across brain tumors lines: poly-L-aspartic acid (semi-synthetic) and hyaluronic acid (natural polysaccharide). The addition of the angiopep-2 targeting moiety onto these polymers improved nanoparticle uptake into brain microvascular endothelial cells in vitro without abrogating tumor affinity. We developed a new algorithm to quantify permeability of fluorescent compounds across the BBB in vivo and validated our method by measuring dextran permeability at varied molecular weights. In our initial study in non-tumor-bearing mice (n=12), we successfully quantified nanoparticle permeability across the BBB. In this study, surface functionalization did not increase BBB permeability above the control nanoparticle, though it did improve nanoparticle half-life in circulation and may still impart a therapeutic benefit when loaded with drug. Additional investigations in orthotopic tumor-bearing mice are ongoing. In summary, we report the development of layer-by-layer nanocarriers as a modular drug delivery platform with therapeutic potential for gliomas.

Vascular Endothelial Glycocalyx Plays a Role in the Obesity Paradox According to Intravital Observation

According to the “obesity paradox,” for severe conditions, individuals with obesity may be associated with a higher survival rate than those who are lean. However, the physiological basis underlying the mechanism of the obesity paradox remains unknown. We hypothesize that the glycocalyx in obese mice is thicker and more resistant to inflammatory stress than that in non-obese mice. In this study, we employed intravital microscopy to elucidate the differences in the vascular endothelial glycocalyx among three groups of mice fed diets with different fat concentrations. Male C57BL/6N mice were divided into three diet groups: low-fat (fat: 10% kcal), medium-fat (fat: 45% kcal), and high-fat (fat: 60% kcal) diet groups. Mice were fed the respective diet from 3 weeks of age, and a chronic cranial window was installed at 8 weeks of age. At 9 weeks of age, fluorescein isothiocyanate-labeled wheat germ agglutinin was injected to identify the glycocalyx layer, and brain pial microcirculation was observed within the cranial windows. We randomly selected arterioles of diameter 15–45 μm and captured images. The mean index of the endothelial glycocalyx was calculated using image analysis and defined as the glycocalyx index. The glycocalyx indexes of the high-fat and medium-fat diet groups were significantly higher than those of the low-fat diet group (p < 0.05). There was a stronger positive correlation between vessel diameter and glycocalyx indexes in the high-fat and medium-fat diet groups than in the low-fat diet group. The glycocalyx indexes of the non-sepsis model in the obese groups were higher than those in the control group for all vessel diameters, and the positive correlation was also stronger. These findings indicate that the index of the original glycocalyx may play an important role in the obesity paradox.

An in vivo Calcium Imaging Approach for the Identification of Cell-Type Specific Patterns in the Developing Cortex

Neuronal activity profoundly shapes the maturation of developing neurons. However, technical limitations have hampered the ability to capture the progression of activity patterns in genetically defined neuronal populations. This task is particularly daunting given the substantial diversity of pyramidal cells and interneurons in the neocortex. A hallmark in the development of this neuronal diversity is the participation in network activity that regulates circuit assembly. Here, we describe detailed methodology on imaging neuronal cohorts longitudinally throughout postnatal stages in the mouse somatosensory cortex. To capture neuronal activity, we expressed the genetically encoded calcium sensor GCaMP6s in three distinct interneuron populations, the 5HT3aR-expressing layer 1 (L1) interneurons, SST interneurons, and VIP interneurons. We performed cranial window surgeries as early as postnatal day (P) 5 and imaged the same cohort of neurons in un-anesthetized mice from P6 to P36. This Longitudinal two-photon imaging preparation allows the activity of single neurons to be tracked throughout development as well as plasticity induced by sensory experience and learning, opening up avenues of research to answer fundamental questions in neural development in vivo.

Abstract P434: A Mouse Model To Control Vessel Diameter With Light

In diseases such as stroke, hypertension, vascular cognitive impairment, and Alzheimer’s disease, defects in the cerebrovascular system lead to reduced blood flow and vasoreactivity to stimuli. Recently, there has been increased appreciation for the role of small vessels in these vascular pathologies. For example, small vessel dysfunction can cause widespread microinfarcts and capillary stalling, which may underlie cognitive impairment in cases where large scale vascular abnormalities are not readily detected. However, vascular function is difficult to dissociate from concurrent neuronal deficits cause by damage to neuronal circuitry in brain pathology. Thus the ability to directly probe smooth muscle contraction of small, individual vessels in the intact brain would be a valuable tool for increasing our understanding of vascular contributions to cognitive impairment. We developed an experimental paradigm to optically probe the contractile function of arterioles in vivo with high spatiotemporal precision. This was done by expressing the excitatory opsin ReaChR in vascular smooth muscle cells and pericytes. Using a 594 nm light-emitting diode we were able to evoke widespread vasoconstriction across the cranial window. With a 1040 nm focused, pulsed laser for two-photon stimulation, we were able to evoke highly localized constrictions targeted to individual pial artery branches or penetrating arterioles. Our dual light-path imaging system allowed the optogenetic stimulation to be performed with simultaneous two-photon imaging to monitor vessel activity. Using a spatial light modulator, we were also able to constrict vessels both above and below the imaging plane. This is a powerful tool to assay vasoconstrictive function of single arterioles across 3-dimensional vascular networks in vivo. It also presents novel opportunities to study conductance of vascular signals and to modulate dynamics of functional hyperemia.