Activity-dependent synaptic plasticity is critical for learning and memory. Considerable attention has been paid to mechanisms that increase or decrease synaptic efficacy, referred to as long-term potentiation (LTP) and long-term depression (LTD), respectively. It is becoming apparent that synaptic activity also modulates the ability to elicit subsequent synaptic changes. We provide direct experimental evidence that this modulation is attributable, at least in part, to variations in the level of postsynaptic depolarization required for inducing plasticity. In slices from adult hippocampal CA1, a brief pairing protocol known to produce LTP can also induce LTD. The voltage-response function for the induction of LTD and LTP in naive synapses exhibits three parts: at a postsynaptic membrane potential during pairing (V(m)) -20 mV, LTP is generated. This function varies with initial synaptic efficacy. In depressed synapses, Theta(-), the V(m) above which LTD is generated, is shifted toward more depolarized V(ms) and Theta(+), the LTD-LTP crossover point or, equivalently, the V(m) above which LTP is induced, toward more polarized V(ms). Conversely in potentiated synapses, Theta(-) is shifted toward more polarized V(ms). Therefore synaptic activity changes synaptic efficacy and accordingly adjusts the voltages for eliciting subsequent synaptic modifications. The concomitant shifts in the voltages for inducing LTD and LTP in opposite directions promote synaptic potentiation and inhibit synaptic depression in depressed synapses and vice versa in potentiated synapses.

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations.

We report on recent advances in the fabrication of three-dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two-photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer-designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient-specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold-like structures, using the photosensitive organic-inorganic hybrid polymer ORMOCER (ORganically MOdified CERamics) and epoxy-based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR-17 granulosa cells was not affected by the presence of ORMOCER. Furthermore, gap junction conductance measurements revealed that ORMOCER did not alter the formation of cell-cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

No abstract