Our finding that PNN digestion is not involved in the observed effects offers an alternative mechanism for this therapeutic strategy in the treatment of CNS trauma

Our finding that PNN digestion is not involved in the observed effects offers an alternative mechanism for this therapeutic strategy in the treatment of CNS trauma. Footnotes This work was supported by Internationale Stiftung fr Forschung in Paraplegie, Zrich, Grant P91; The Christopher and Dana Reeve Foundation; and The Isaac Newton Trust of the University of Cambridge. blocking antibody. Interestingly, microinjection of ChABC close to dendritic segments was sufficient to induce spine remodeling, demonstrating that CSPGs located around dendritic spines modulate PTK2 their dynamics independently of perineuronal nets. This restrictive action of perisynaptic CSPGs in mature neural tissue may account for the therapeutic effects of ChABC in promoting functional recovery in impaired neural circuits. Introduction The early postnatal development of the CNS is characterized by a critical period for plasticity during which circuits are shaped and connections refined in an experience-dependent manner (Knudsen, 2004; Hensch, 2005). This plasticity is associated with dynamic processes involving the formation, elimination, and remodeling of dendritic spines, the sites of excitatory synaptic connections. In rodents, the critical period for plasticity closes early after birth and marks a decline in spines dynamics. In parallel, the juvenile type of extracellular matrix (ECM) is replaced by its mature form that persists throughout adulthood (Frischknecht and Gundelfinger, 2012). Proteolysis of the mature ECM restores spine plasticity suggesting a role for the ECM in stabilizing dendritic spines. Chondroitin sulfate proteoglycans (CSPGs) are the main components of the mature ECM. As the critical period comes to an end, these proteins undergo changes in their core composition, in sulfation patterns and GS967 in distribution (Deepa et al., 2006; Miyata et al., 2012), forming a diffused ubiquitous matrix as well as dense perineuronal nets (PNNs) mainly surrounding parvalbumin-expressing fast-spiking interneurons (H?rtig et al., 1999). digestion of CSPGs mediated by the bacterial enzyme chondroitinase ABC (ChABC) restores functional plasticity in various models of CNS pathologies (Kwok et al., 2011); however, the mechanisms mediating these effects are still poorly understood. In particular, while most attention has been focused on PNNs, the role of the diffused ECM in controlling CNS structural plasticity has been so far neglected. Nevertheless, the ECM that fills perisynaptic spaces surrounding dendritic spines may play an important role in restricting the remodeling of neuronal circuits at the synaptic level. We explored the effects of ChABC-mediated CSPG digestion on dendritic spine dynamics by performing live imaging in mature hippocampal slice cultures. We could show that ChABC treatment has effects independent of PNN digestion that lead to enhanced motility of dendritic spines and to the formation of spine head protrusions. These GS967 dynamic changes are driven by 1-integrin activation and phosphorylation of focal adhesion kinase (FAK) at synaptic sites. Materials and Methods Organotypic slice cultures. Organotypic slice cultures of hippocampus were prepared from Thy1-YFP pups (H-line; The Jackson Laboratory) or wild-type Bl/6 at postnatal day 6 as previously described (G?hwiler et al., 1997) and maintained in roller tubes for periods ranging from 1 to 5 weeks. Culture medium (50% Basal Medium Eagle, 25% inactivated horse serum, 25% HBSS, 5.6 mm glucose, and 200 mm l-glutamine) was changed every week. Slices gradually mature to form stable circuits whose development resembles, both in terms of timing and connectivity, the situation (Muller et al., 1993; De Simoni et al., 2003; Cho et al., 2007). This model is well suited GS967 for performing chronic treatments and long-term imaging. Enzymatic treatment. Digestion of CSPGs was achieved by treatment with protease-free ChABC from (Seikagaku). ChABC was GS967 reconstituted in 0.1 m phosphate buffer (PB) (0.1 U/l), pH 7.4, before being added to the culture medium. Slices.

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