MeCP2 deficiency impairs motor cortical circuit flexibility associated with motor learning.

Loss of function mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2) cause Rett syndrome (RTT), a postnatal neurological disorder. The loss of motor function is an important clinical feature of RTT that manifests early during the course of the disease. RTT mouse models with mutations in the murine orthologous Mecp2 gene replicate many human phenotypes, including progressive motor impairments. However, relatively little is known about the changes in circuit function during the progression of motor deficit in this model. As the motor cortex is the key node in the motor system for the control of voluntary movement, we measured firing activity in populations of motor cortical neurons during locomotion on a motorized wheel-treadmill. Different populations of neurons intermingled in the motor cortex signal different aspects of the locomotor state of the animal. The proportion of running selective neurons whose activity positively correlates with locomotion speed gradually decreases with weekly training in wild-type mice, but not in Mecp2-null mice. The fraction of rest-selective neurons whose activity negatively correlates with locomotion speed does not change with training in wild-type mice, but is higher and increases with the progression of locomotion deficit in mutant mice. The synchronization of population activity that occurs in WT mice with training did not occur in Mecp2-null mice, a phenotype most clear during locomotion and observable across all functional cell types. Our results could represent circuit-level biomarkers for motor regression in Rett syndrome.

Pattern decorrelation in the mouse medial prefrontal cortex enables social preference and requires MeCP2.

Sociability is crucial for survival, whereas social avoidance is a feature of disorders such as Rett syndrome, which is caused by loss-of-function mutations in MECP2. To understand how a preference for social interactions is encoded, we used in vivo calcium imaging to compare medial prefrontal cortex (mPFC) activity in female wild-type and Mecp2-heterozygous mice during three-chamber tests. We found that mPFC pyramidal neurons in Mecp2-deficient mice are hypo-responsive to both social and nonsocial stimuli. Hypothesizing that this limited dynamic range restricts the circuit's ability to disambiguate coactivity patterns for different stimuli, we suppressed the mPFC in wild-type mice and found that this eliminated both pattern decorrelation and social preference. Conversely, stimulating the mPFC in MeCP2-deficient mice restored social preference, but only if it was sufficient to restore pattern decorrelation. A loss of social preference could thus indicate impaired pattern decorrelation rather than true social avoidance.

Deep-learning two-photon fiberscopy for video-rate brain imaging in freely-behaving mice.

Scanning two-photon (2P) fiberscopes (also termed endomicroscopes) have the potential to transform our understanding of how discrete neural activity patterns result in distinct behaviors, as they are capable of high resolution, sub cellular imaging yet small and light enough to allow free movement of mice. However, their acquisition speed is currently suboptimal, due to opto-mechanical size and weight constraints. Here we demonstrate significant advances in 2P fiberscopy that allow high resolution imaging at high speeds (26 fps) in freely-behaving mice. A high-speed scanner and a down-sampling scheme are developed to boost imaging speed, and a deep learning (DL) algorithm is introduced to recover image quality. For the DL algorithm, a two-stage learning transfer strategy is established to generate proper training datasets for enhancing the quality of in vivo images. Implementation enables video-rate imaging at ~26 fps, representing 10-fold improvement in imaging speed over the previous 2P fiberscopy technology while maintaining a high signal-to-noise ratio and imaging resolution. This DL-assisted 2P fiberscope is capable of imaging the arousal-induced activity changes in populations of layer2/3 pyramidal neurons in the primary motor cortex of freely-behaving mice, providing opportunities to define the neural basis of behavior.

Motor training improves coordination and anxiety in symptomatic Mecp2-null mice despite impaired functional connectivity within the motor circuit.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by loss of function of the X-linked methyl-CpG­binding protein 2 (MECP2). Several case studies report that gross motor function can be improved in children with RTT through treadmill walking, but whether the MeCP2-deficient motor circuit can support actual motor learning remains unclear. We used two-photon calcium imaging to simultaneously observe layer (L) 2/3 and L5a excitatory neuronal activity in the motor cortex (M1) while mice adapted to changing speeds on a computerized running wheel. Despite circuit hypoactivity and weakened functional connectivity across L2/3 and L5a, the Mecp2-null circuit's firing pattern evolved with improved performance over 2 weeks. Moreover, trained mice became less anxious and lived 20% longer than untrained mice. Because motor deficits and anxiety are core symptoms of RTT, which is not diagnosed until well after symptom onset, these results underscore the benefit of motor learning.

Throughput-Speed Product Augmentation for Scanning Fiber-Optic Two-Photon Endomicroscopy.

Compactness, among several others, is one unique and very attractive feature of a scanning fiber-optic two-photon endomicroscope. To increase the scanning area and the total number of resolvable pixels (i.e., the imaging throughput), it typically requires a longer cantilever which, however, leads to a much undesired, reduced scanning speed (and thus imaging frame rate). Herein we introduce a new design strategy for a fiber-optic scanning endomicroscope, where the overall numerical aperture (NA) or beam focusing power is distributed over two stages: 1) a mode-field focuser engineered at the tip of a double-clad fiber (DCF) cantilever to pre-amplify the single-mode core NA, and 2) a micro objective of a lower magnification (i.e.,  âˆ¼ 2× in this design) to achieve final tight beam focusing. This new design enables either an ~9-fold increase in imaging area (throughput) or an ~3-fold improvement in imaging frame rate when compared to traditional fiber-optic endomicroscope designs. The performance of an as-designed endomicroscope of an enhanced throughput-speed product was demonstrated by two representative applications: (1) high-resolution imaging of an internal organ (i.e., mouse kidney) in vivo over a large field of view without using any fluorescent contrast agents, and (2) real-time neural imaging by visualizing dendritic calcium dynamics in vivo with sub-second temporal resolution in GCaMP6m-expressing mouse brain. This cascaded NA amplification strategy is universal and can be readily adapted to other types of fiber-optic scanners in compact linear or nonlinear endomicroscopes.

High-speed fiber-optic scanning nonlinear endomicroscopy for imaging neuron dynamicsin vivo.

Fiber-optic-based two-photon fluorescence endomicroscopy is emerging as an enabling technology for in vivo histological imaging of internal organs and functional neuronal imaging on freely-behaving animals. However, high-speed imaging remains challenging due to the expense of miniaturization and lack of suited fast beam scanners. For many applications, a higher imaging speed is highly desired, especially for monitoring functional dynamics such as transient dendritic responses in neuroscience. This Letter reports the development of a fast fiber-optic scanning endo-microscope with an imaging speed higher than 26 frames/s. In vivo neural dynamics imaging with the high-speed endomicroscope was performed on a freely-behaving mouse over the primary motor cortex that expressed GCaMP6m. The results demonstrate its capability of real-time monitoring of transient neuronal dynamics with very fine temporal resolution.

In Vivo Imaging Reveals Composite Coding for Diagonal Motion in the Drosophila Visual System.

Understanding information coding is important for resolving the functions of visual neural circuits. The motion vision system is a classic model for studying information coding as it contains a concise and complete information-processing circuit. In Drosophila, the axon terminals of motion-detection neurons (T4 and T5) project to the lobula plate, which comprises four regions that respond to the four cardinal directions of motion. The lobula plate thus represents a topographic map on a transverse plane. This enables us to study the coding of diagonal motion by investigating its response pattern. By using in vivo two-photon calcium imaging, we found that the axon terminals of T4 and T5 cells in the lobula plate were activated during diagonal motion. Further experiments showed that the response to diagonal motion is distributed over the following two regions compared to the cardinal directions of motion-a diagonal motion selective response region and a non-selective response region-which overlap with the response regions of the two vector-correlated cardinal directions of motion. Interestingly, the sizes of the non-selective response regions are linearly correlated with the angle of the diagonal motion. These results revealed that the Drosophila visual system employs a composite coding for diagonal motion that includes both independent coding and vector decomposition coding.

1064 nm-Nd:YAG lasers with different output modes enhancing transdermal delivery: physical and physiological mechanisms.

Lasers have shown great advantages in enhancing transdermal drug delivery. However, the physical or physiological mechanisms are not clear, which limits the application in clinical medicine. Here, 1064 nm-Nd:YAG lasers with long-pulsed (LP, 15 J/cm²) and Q-switched (QS, 0.5 J/cm²) output modes inducing short- and long-term effects on the stratum corneum (SC) of skin are investigated. Infrared thermography is applied to monitor the dynamical temperature distribution of the skin surface, while histopathological analysis and two-photon fluorescence microscopy are employed to examine changes in the microstructure of skin and molecular constitution of SC, respectively. Results have shown that the LP laser irradiation increases skin temperature evidently and loosens keratin, making corneocytes fragile or exfoliative, whereas the QS laser irradiation disrupts the keratin or corneocytes completely, perforating some micropores on the SC. It can be concluded that the mechanisms of enhancing transdermal delivery caused by lasers depends on the output modes. The LP laser irradiation produces thermal effects on skin, which loosens the SC, while the QS laser induces mechanical effects on skin, which punches micropores on the SC. Moreover, the laser-induced enhancing effects on transdermal glycerol delivery can last for one week to wait for the recovery of SC.