Understanding the endogenous repair capacity of spinal cord is pivotal to develop strategies to improve it. Here we design a paradigm of spinal cord lesion in the dorsal column using a 2-photon microscopy technique to dynamically and chronically monitor simultaneous changes of vascular and axonal networks in living mice up to 4 months postinjury. High-resolution images showed that early explorative sprouting of surviving injured axons resulted in extensive regrowth until and past the lesion site within 2 months. Blood vessel density was transiently up-regulated and most neurovascular interactions occurred within 2 weeks. Timelapse analysis showed that neovessels exerted a potent growth stimulating action, but no guidance effect on neighboring sprouts, possibly because of their geometry and plasticity. Nevertheless, if reconnection depends on axon sprout density, stimulation of angiogenesis would probably be beneficial to repair. More generally, this imaging approach is showing promise to aid in monitoring brain diseases and the efficacy of potential treatments.angiogenesis ͉ axon regeneration ͉ in vivo 2-photon imaging ͉ spinal cord injury ͉ time lapse S pinal cord injury (SCI) induces dramatic cellular and molecular changes in the neuronal environment resulting overall in impaired axonal regeneration (1-3). Posttraumatic disturbance of blood perfusion is, for example, responsible for secondary damage (3), and reports suggest that stimulation of posttraumatic angiogenesis with vascular endothelial growth factor (VEGF) improves functional recovery of injured rodents (4, 5). Optimizing treatments to target vascularization might then be a valuable therapeutic strategy in the context of SCI, but the finding that VEGF signals on axons in addition to blood vessels (6) challenges the importance of the vascular response in the recovery process.The conclusion of these studies is that the dynamics of the interactions occurring between axons and blood vessels and the influence of vascularization on axon regrowth have to be characterized thoroughly at the single axon level. Investigative tools are scarce, however, and poorly compatible with the determination of kinetics as most of the acquired knowledge comes from histological studies on spinal cord sections sampled from different subjects at different time points. A pioneering in vivo study combining wide-field imaging and the use of transgenic mice with fluorescent spinal cord axons reported observations of individual axons over 1 week after SCI (7). However, only a few axons were sampled per mouse and they were observed for a limited time. We sought to design a more dedicated imaging protocol on the basis of 2-photon microscopy (8, 9) to resolve populations of injured axons in 3 dimensions (3D) in the same mouse and to optimize the method to allow dynamic studies over a period of several months. Dual-color imaging allowed simultaneous visualization of posttraumatic vascular responses and for the first time neurovascular interactions were characterized in vivo at a cellul...

The thalamic reticular nucleus (RTN) is the major source of inhibitory contacts in the thalamus and thus plays an important role in regulating the excitability of the thalamocortical network. Inhibition occurs through GABAergic synapses on relay cells as well as through GABAergic synapses between reticularis neurons themselves. Here we report that the role and mechanisms of this inhibition, which frequently have been studied using N-methyl derivatives of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline, should be revisited. Using the whole cell patch-clamp technique in thalamic slices from young rats, we observed an enhancement by bicuculline methiodide, methobromide, and methochloride (collectively referred to as bicuculline-M; 5-60 microM) of the low-threshold calcium spike burst in RTN neurons that persisted in the presence of tetrodotoxin (1 microM) and was not reproduced in picrotoxin (100-300 microM). The effect did not involve activation of any GABA receptor subtype. Voltage-clamp recordings showed that bicuculline-M blocked the current underlying the low-threshold spike burst afterhyperpolarization (AHP), an effect that was mimicked by apamin (100 nM). Recordings from nucleated patches extracted from reticularis neurons demonstrated that this effect was not mediated by modulation of the release of an unidentified neurotransmitter but that bicuculline-M directly blocks small conductance (SK) channels. The AHP-blocking effect also was observed in other brain regions, demonstrating that although bicuculline-M is a potent GABAA receptor antagonist, it is of limited value in assessing GABAergic network interactions, which should be studied using picrotoxin or bicuculline-free base. However, bicuculline-M may provide a useful tool for developing nonpeptide antagonists of SK channels.

Glioblastoma multiforme (GBM) are highly invasive and angiogenic malignancies with a median survival time from diagnosis of <15 months. Previous work has revealed robust overexpression of fibronectin (FN) mRNA in GBM, although immunohistochemical staining of FN in these tumors is typically associated with the angiogenic vasculature. Here we sought to examine the expression of tumor cell FN and address its possible involvement in the invasive phenotype of GBM. We found that FN was expressed and assembled into fibrillar arrays in human tumors and in established GBM lines. Cultured cells spontaneously formed dense cellular networks and spheroid-like domes. Depletion of FN by targeted-short hairpin RNA expression disrupted matrix assembly and multicellular network organization by exerting profound effects on cell adhesion and motility. Although FN depletion enhanced persistent directional migration of single cells, it compromised collective invasion of spheroids through a laminin-rich matrix and sensitized cells to ionizing radiation. In orthotopic grafts, FN depletion significantly reduced tumor growth and angiogenesis. Together our results show that FN produced by the tumor cells has a role in GBM pathophysiology and they provide insights into the implications that targeting FN interactions may have for combating this dreaded disease.

Drug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments.