A small-molecule TrkB ligand improves dendritic spine phenotypes and atypical behaviors in female Rett syndrome mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein-2 (MECP2), encoding a transcriptional regulator of many genes, including brain-derived neurotrophic factor (Bdnf). BDNF mRNA and protein levels are lower in RTT autopsy brains and in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of a brain penetrant, small molecule ligand of its TrkB receptors. Applied in vitro, LM22A-4 increased dendritic spine density in pyramidal neurons in cultured hippocampal slices from postnatal day (P) 7 male Mecp2 knockout (KO) mice as much as recombinant BDNF, and both effects were prevented by the TrkB receptor inhibitors K-252a and ANA-12. Consistent with its partial agonist activity, LM22A-4 did not affect spine density in CA1 pyramidal neurons in slice cultures from male wildtype (WT) mice, where typical BDNF levels outcompete its binding to TrkB. To identify neurons of known genotypes in the "mosaic" brain of female Mecp2 heterozygous (HET) mice, we treated 4-6-month-old female MeCP2-GFP WT and HET mice with peripheral injections of LM22A-4 for 4 weeks. Surprisingly, mutant neurons lacking MeCP2-GFP showed dendritic spine volumes comparable to that in WT controls, while MeCP2-GFP-expressing neurons showed larger spines, similar to the phenotype we described in symptomatic male Mecp2 KO mice where all neurons lack MeCP2. Consistent with this non-cell-autonomous mechanism, a 4-week systemic treatment with LM22A-4 had an effect only in MeCP2-GFP-expressing neurons in female Mecp2 HET mice, bringing dendritic spine volumes down to WT control levels, and without affecting spines of MeCP2-GFP-lacking neurons. At the behavioral level, we found that female Mecp2 HET mice engaged in aggressive behaviors significantly more than WT controls, which were reduced to WT levels by a 4-week systemic treatment with LM22A-4. Altogether, these data revealed differences in dendritic spine size and altered behaviors in Mecp2 HET mice, while providing support to the potential usefulness of BDNF-related therapeutic approaches such as the partial TrkB agonist LM22A-4.

The role of MeCP2 in learning and memory.

Gene transcription is a crucial step in the sequence of molecular, synaptic, cellular, and systems mechanisms underlying learning and memory. Here, we review the experimental evidence demonstrating that alterations in the levels and functionality of the methylated DNA-binding transcriptional regulator MeCP2 are implicated in the learning and memory deficits present in mouse models of Rett syndrome and MECP2 duplication syndrome. The significant impact that MeCP2 has on gene transcription through a variety of mechanisms, combined with well-defined models of learning and memory, make MeCP2 an excellent candidate to exemplify the role of gene transcription in learning and memory. Together, these studies have strengthened the concept that precise control of activity-dependent gene transcription is a fundamental mechanism that ensures long-term adaptive behaviors necessary for the survival of individuals interacting with their congeners in an ever-changing environment.

Non-invasive optical imaging of the lymphatic vasculature of a mouse.

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging using indocyanine green (ICG), a dye that has been used for 50 years in humans, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 µl) of ICG (645 µM) or cABD-IRDye800 (200 µM) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 µg/kg for ICG or 113-1,700 µg/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.