Dual simulated childbirth injury delays anatomic recovery

A dual childbirth injury model, including vaginal distension (VD) and pudendal nerve crush (PNC), may best represent the injuries seen clinically. The objective of this study was to investigate urethral function, anatomy, and neurotrophin expression after several simulated childbirth injuries. Groups of 140 rats underwent PNC, VD, PNC+VD, or neither (C). Four days after injury, all injury groups had significantly decreased leak-point pressure (LPP) compared with C rats. Ten days after injury, LPP in PNC and PNC+VD rats remained significantly lower than C rats. Three weeks after injury, LPP in all injury groups had recovered to C values. Histological evidence of injury was still evident in the external urethral sphincter (EUS) after VD and PNC+VD 10 days after injury. Three weeks after injury, the EUS of PNC+VD rats remained disrupted. One day after VD, brain-derived neurotrophic factor (BDNF) expression in the EUS was reduced, while neurotrophin-4 (NT-4) and nerve growth factor (NGF) expression was unchanged. BDNF, NT-4, and NGF expression was dramatically upregulated in the EUS after PNC. After PNC+VD, NGF expression was upregulated, and BDNF and NT-4 expression was upregulated somewhat but not to the same extent as after PNC. Ten days after injury, PNC+VD had the least number of normal nerve fascicles near the EUS, followed by PNC and VD. Twenty-one days after injury, all injury groups had fewer normal nerve fascicles, but without significant differences compared with C rats. PNC+VD therefore provides a more severe injury than PNC or VD alone

Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force “barrier” on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRA∶B linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation

Interaction of nucleus reuniens and entorhinal cortex projections in hippocampal field CA1 of the rat

The nucleus reuniens (RE) and entorhinal cortex (EC) provide monosynaptic excitatory inputs to the apical dendrites of pyramidal cells and to interneurons with dendrites in stratum lacunosum moleculare (LM) of hippocampal field CA1. However, whether the RE and EC inputs interact at the cellular level is unknown. In this electrophysiological in vivo study, low-frequency stimulation was used to selectively activate each projection at its origin; field excitatory postsynaptic potentials (fEPSPs) were recorded in CA1. We applied (1) paired pulses to RE or EC, (2) combined paired pulses to RE and EC, and (3) simultaneously paired pulses to RE/EC. The main findings are that: (a) stimulation of either RE- or EC-evoked subthreshold fEPSPs, displaying paired pulse facilitation (PPF), (b) subthreshold fEPSPs evoked by combined stimulation did not display heterosynaptic PPF, and (c) simultaneous stimulation of RE/EC resulted in enhanced subthreshold fEPSPs in proximal LM displaying a nonlinear interaction. CSD analyses of RE/EC-evoked depth profiles revealed a nonlinear enlargement of the 'LM sink-radiatum source' configuration and the appearance of an additional small sink-source pair close to stratum pyramidale, likely reflecting (peri)somatic inhibition. The nonlinear interaction between both inputs indicates that RE and EC axons form synapses, at least partly, onto the same dendritic compartments of CA1 pyramidal cells. We propose that low-frequency activation of the RE-CA1 input facilitates the entorhinal-hippocampal dialogue, and may synchronize the neocortical-hippocampal slow oscillation which is relevant for hippocampal-dependent memory consolidation