The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Journal of Physiology Physiology in PressRBCs in the rat. We first assessed the spatial distribution of glycine receptors on RBCs by direct patch-clamp recordings of isolated presynaptic terminals and by focal puffing of glycine in retinal slices. We found that glycine receptors are highly concentrated at the axon terminals of RBCs. The pharmacological and biophysical properties of glycine receptors located in the axon terminal and somatic/dendritic regions are similar. Furthermore, we present evidence for the existence of glycinergic synaptic inputs onto the axon terminals of RBCs and show that activation of glycine receptors could effectively suppress depolarization-evoked calcium influx into the axon terminals. METHODS Dissociation of bipolar cellsBipolar cells were dissociated from Long-Evans rats ≤4 weeks of age as described previously Pan, 2000). All animal handling procedures were approved by the Institutional Animal Care Committee at Wayne State University, and were in accord with the NIH Guide for the Care and Use of Laboratory Animals. In brief, animals were deeply anaesthetized with CO 2 and killed by decapitation. Retinas were removed and placed in a Hanks' solution (mM): NaCl, 138; NaHCO 3 , 1; Na 2 HPO 4 , 0.3; KCl, 5; KH 2 PO 4 , 0.3; CaCl 2 , 1.25 or 2.5; MgSO 4 , 0.5; MgCl 2 , 0.5; Hepes-NaOH, 5; glucose, 22.2; with phenol red, 0.001 % v/v; pH 7.2. The retinas were incubated for ~40-50 min at 34-37°C in an enzymatic solution that consisted of Hanks' solution (described above), supplemented with DL-cysteine, 0.2 mg ml _1 ; bovine serum albumin, 0.2 mg ml _1 ; and papain, ~2 u ml _1 . After several rinses in Hanks' solution, the retinas were mechanically dissociated by gentle trituration with a glass pipette. The dissociated cells were plated onto culture dishes in normal or Ca 2+ -free Hanks' solution. Cells were kept at room temperature and used for recordings within 5 h. The use of Ca 2+ -free Hanks' solution for cell culture was found to help maintain healthier axon terminals of bipolar cells. The Ca 2+ -free Hanks' solution is the same as the Hanks' solution described above except for the omission of Ca 2+ ions.

Retinal bipolar cells comprise multiple subtypes that are well known for the diversity of their physiological properties. We investigated the properties and functional roles of the hyperpolarization-activated currents in mammalian retinal bipolar cells using whole cell patch-clamp recording techniques. We report that bipolar cells express inwardly rectifying K+ currents ( IKir) in addition to the hyperpolarization-activated cationic currents ( Ih) previously reported. Furthermore, these two currents are differentially expressed among different subtypes of bipolar cells. One group of cone bipolar cells in particular displayed mainly IKir. A second group of cone bipolar cells displayed both currents but with a much larger Ih. Rod bipolar cells, on the other hand, showed primarily Ih. Moreover, we showed that IKir and Ih differentially influence the voltage responses of bipolar cells: Ih facilitates and/or accelerates the membrane potential rebound, whereas IKir counteracts or prevents such rebound. The findings of the expression of IKir and the differential expression of Ih and IKir in bipolar cells may provide new insights into an understanding of the physiological properties of bipolar cells.

Patch-clamp recordings were used to investigate the properties of the regenerative activity in acutely isolated bipolar cells from the rat retina. Spontaneous, pacemaker-like membrane potential oscillations were observed in all rod bipolar cells and the majority of cone bipolar cells. The waveform of the regenerative potential was stereotypical but distinct among different bipolar cell groups, especially between rod and cone bipolar cells. The spontaneous activity was completely blocked by Co2+, suggesting that Ca2+ influx through voltage-dependent Ca2+ channels was required for initiating such activity. Ca2+-induced Ca2+ release, however, was not found to be involved. The spontaneous activity was also blocked by mibefradil, a T-type Ca2+ channel antagonist. In contrast, application of nimodipine, an L-type Ca2+ current antagonist, affected mainly the waveform of the regenerative potential. This study shows that mammalian retinal bipolar cells in isolation are also capable of generating Ca2+-dependent spontaneous regenerative potential. However, T-type Ca2+ channels appear to be essential for the initiation of the spontaneous activity in mammalian bipolar cells.

Retinal bipolar cells show heterogeneous expression of voltage-dependent Na+ and K+ currents. We used whole-cell patch-clamp recordings to investigate the possible roles of these currents in the response properties of bipolar cells in rats. Isolated bipolar cells showed robust spontaneous regenerative activity, but the regenerative potential of rod bipolar cells reached a more depolarized level than that of cone bipolar cells. In both isolated cells and cells in retinal slices, the membrane depolarization evoked by current injection was apparently capped. The evoked membrane potential was again more depolarized in rod bipolar cells than in cone bipolar cells. Application of tetraethylammonium and 4-aminopyridine shifted the spontaneous regenerative potential as well as the evoked potential to a more depolarized level. In addition, a subclass of cone bipolar cells showed a prominent spike in the initial phase of the voltage response when the cells were depolarized from a relatively negative membrane potential. The spike was mediated mainly by tetrodotoxin-sensitive Na+ current. The presence of the spike sped up the response kinetics and enhanced the peak membrane potential. Results of this study raise the possibility that voltage-dependent K+ currents may play a role in defining different membrane operating ranges of rod and cone bipolar cells and that voltage-dependent Na+ currents may enhance the response kinetics and amplitude of certain cone bipolar cells.

Application of synthetic or biological meshes is the main therapy for the repair and reconstruction of abdominal wall defects, a common disease in surgery. Currently, no ideal materials are available, and there is an urgent need to find appropriate ones to satisfy clinical needs. Electrospun scaffolds have drawn attention in soft tissue reconstruction. In this study, we developed a novel method to fabricate a composite electrospun scaffold using a thermoresponsive hydrogel, poly (N-isopropylacrylamide)-block-poly (ethylene glycol), and a biodegradable polymer, polylactic acid (PLA). This scaffold provided not only a high surface area/volume ratio and a three-dimensional fibrous matrix but also high biocompatibility and sufficient mechanical strength, and could simulate the native extracellular matrix and accelerate cell adhesion and proliferation. Furthermore, rat adipose-derived stem cells (ADSCs) were seeded in the composite electrospun scaffold to enhance the defect repair and regeneration by directionally inducing ADSCs into endothelial cells. In addition, we found early vascularization in the process was regulated by the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway. In our study, overexpression of HIF-1α/VEGF in ADSCs using a lentivirus system promoted early vascularization in the electrospun scaffolds. Overall, we expect our composite biomimetic scaffold method will be applicable and useful in abdominal wall defect regeneration and repair in the future.

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