Characterization of myelin degradation in the white matter (WM) is important for understanding neurodegeneration. We demonstrate label-free in vivo imaging of myelin structure in the WM of mice, through intact cortex, using third harmonic generation (THG) microscopy at 1320-nm excitation. Longitudinal THG imaging of the same axons in the cuprizone mouse model of multiple sclerosis revealed dynamics of myelin blistering. Further, we measured intranodal distance at nodes of Ranvier in vivo and developed a novel metric of myelin structural change based on spatial concentration of the brightest THG signal. We also demonstrated compatibility with three-photon excited fluorescence microscopy by imaging GFP-labeled microglia in the WM in parallel with THG microscopy, thereby enabling detailed tracking of subcortical myelin and other cellular dynamics in neurodegenerative disease models.

Transient stoppages of red blood cell (RBC) flow through capillaries—termed capillary stalls—occur persistently in neurological disorders such as Alzheimer’s disease and ischemic stroke and can interrupt oxygen delivery and exacerbate neurological damage. Effective imaging tools and analyses are necessary to understand the nature, role, and prevention of stalls. In this study, we dissect differences in stalls measured by two-photon Bessel beam microscopy (Bessel-2PM) and optical coherence tomography (OCT) to gain insight into the temporal dynamics of stalls. Twenty-minute series of volumetric angiograms were obtained separately with Bessel-2PM and OCT on the same day in awake, head-fixed mice. The temporal dynamics of stalling in both methods revealed a minority population of susceptible capillaries that exhibited frequent stalls and a large majority of capillaries with infrequent stalls. Differences between OCT and Bessel-2PM in the repeatability and dynamics of stalls are explained by differences in their sensitivity to short or infrequent stalls based on scanning speed and detection off-time. Finally, stroke caused a shift toward the frequently stalling capillary subpopulation, lasting 1 week post-stroke. Dynamic stall analysis therefore enables examination of physiological and methodological contributions to the stalls measured in disease models and across studies.

In partial onset epilepsy, seizures arise focally in the brain and often propagate. Patients frequently become refractory to medical management, leaving neurosurgery, which can cause neurologic deficits, as a primary treatment. In the cortex, focal seizures spread through horizontal connections in layers II/III, suggesting that severing these connections can block seizures while preserving function. Focal neocortical epilepsy is induced in mice, sub-surface cuts are created surrounding the seizure focus using tightly-focused femtosecond laser pulses, and electrophysiological recordings are acquired at multiple locations for 3–12 months. Cuts reduced seizure frequency in most animals by 87%, and only 5% of remaining seizures propagated to the distant electrodes, compared to 80% in control animals. These cuts produced a modest decrease in cortical blood flow that recovered and left a ~20-µm wide scar with minimal collateral damage. When placed over the motor cortex, cuts do not cause notable deficits in a skilled reaching task, suggesting they hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy.

The blood–brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5’-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Advanced imaging shows the mouse heart as it pumps, leading to insights into cardiac physiology and disease genesis.

Small-animal virtual reality (VR) systems have become invaluable tools in neuroscience for studying complex behavior during head-fixed neural recording, but they lag behind commercial human VR systems in terms of miniaturization, immersivity and advanced features such as eye tracking. Here we present MouseGoggles, a miniature VR headset for head-fixed mice that delivers independent, binocular visual stimulation over a wide field of view while enabling eye tracking and pupillometry in VR. Neural recordings in the visual cortex validate the quality of image presentation, while hippocampal recordings, associative reward learning and innate fear responses to virtual looming stimuli demonstrate an immersive VR experience. Our open-source system’s simplicity and compact size will enable the broader adoption of VR methods in neuroscience.

Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact during imaging or other measurements. Anesthetized animals must be closely monitored for the safety of the animals and to prevent unintended effects of altered physiology on experimental outcomes. Many currently available monitoring devices are expensive, invasive, or interfere with experimental design. Here, we present MousePZT, a low-cost device based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate matches those of commercial systems. Using the widely-used gas isoflurane and injectable ketamine/xylazine combination, we also demonstrate that changes in respiratory rate are more easily detected and can precede changes in heart rate associated with variations in anesthetic depth. Additional circuitry on the device outputs a respiration-locked trigger signal for respiratory-gating of imaging or other data acquisition and has high sensitivity and specificity for detecting respiratory cycles. We provide detailed instruction documents and all necessary microcontroller and computer software, enabling straightforward construction and utilization of this device.

CD81 T lymphocytes infiltrate the brain during congenital CMV infection and promote viral clearance. However, the mechanisms by which CD81 T cells are recruited to the brain remain unclear. Using a mouse model of congenital CMV, we found a gut-homing chemokine receptor (CCR9) was preferentially expressed in CD81 T cells localized in the brain postinfection. In the absence of CCR9 or CCL25 (CCR9’s ligand) expression, CD81 T cells failed to migrate to key sites of infection in the brain and protect the host from severe forms of disease. Interestingly, we found that expression of CCR9 on CD81 T cells was also responsible for spatial temporal positioning of T cells in the brain. Collectively, our data demonstrate that the CMVinfected brain uses a similar mechanism for CD81 T cell homing as the small intestine.

Two-photon excited fluorescence microscopy is a widely-employed imaging technique that enables the noninvasive study of biological specimens in three dimensions with submicrometer resolution. Here, we report an assessment of a gain-managed nonlinear (GMN) fiber amplifier for multiphoton microscopy. This recently-developed source delivers 58-nJ and 33-fs pulses at 31-MHz repetition rate. We show that the GMN amplifier enables high-quality deep-tissue imaging, and furthermore that the broad spectral bandwidth of the GMN amplifier can be exploited for superior spectral resolution when imaging multiple distinct fluorophores.

Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.