Synergistic protection of RGCs by olfactory ensheathing cells and alpha-crystallin through regulation of the Akt/BAD Pathway.

BACKGROUND: Our recent in vivo studies have shown that olfactory ensheathing cells (OECs) and α-crystallin can promote retinal ganglion cell (RGC) survival and axonal regeneration synergistically after optic nerve injury. However, the mechanism is still unknown. OBJECTIVES: Here, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGC survival after H2O2-induced oxidative damage and a crushing injury to the optic nerve in an adult rat model. METHODS: After H2O2-induced oxidative damage, RGC-5 cells were treated with OECs, α-crystallin or a combination of OECs and α-crystallin. Apoptosis of RGC-5 cells was assessed by flow cytometry. Phosphorylated Akt, BAD, and cleaved-caspase3 were detected by Western blot after optic nerve injury in vivo and H2O2-induced RGC-5 oxidative damage in vitro. RESULTS: The results showed that OECs and α-crystallin could both independently inhibit RGC-5 apoptosis (P<0.01), increase the phosphorylation of both Akt and BAD, and decrease the activation of caspase-3 (P<0.01). However, the effect of the combination of both was more significant than either alone. CONCLUSION: These findings indicate that inhibition of superoxide damage to RGCs through regulation of the Akt/BAD pathway is one of the mechanisms by which OECs and α-crystallin promote optic nerve recovery after injury.

3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration.

The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

Modeling axon membranes for functional electrical stimulation.

Four models are discussed which can be used to predict the behavior of warm-blooded axons, when excited by electric fields. Up to now, most results were obtained with the Frankenhaeuser-Huxley model, but nearly all of them are wrong in time scale and the cathodic block phenomenon was not observable because the temperature dependence of the gating mechanism has been neglected. However, in the corrected form this model reacts with similar excitability as the two other myelinated nerve models which consider that the potassium current is negligible in mammalian axon membranes. Strength-duration relations for cathodic and anodic excitations, as well as for cathodic blockade, are presented. Paradoxically, the "warm" squid model of Hodgkin and Huxley is the only one which reflects phenomena known from stimulations of the (myelinated) acoustic nerve by cochlear implants.

Membrane and synaptic properties of developing lateral geniculate nucleus neurons during retinogeniculate axon segregation.

During the first postnatal month in the ferret (Mustela putorius furo), the projections from the retina to the lateral geniculate nucleus (LGN) become segregated into eye-specific layers and ON and OFF sublayers, a process that is thought to depend in part on neuronal activity. Remarkably, virtually nothing is known about the physiological features of LGN neurons during this period. We have recorded intracellularly from 46 A-layer neurons in slices of the ferret LGN between the ages of postnatal days 7 and 33. The passive membrane properties and current-voltage relationships of the developing neurons were similar in many, though not all, respects to those of adult LGN neurons. Action potentials in younger animals were smaller in amplitude and longer in duration than in older animals, but cells at all ages were capable of producing spike trains whose latency and spike number varied with stimulus intensity. In addition, cells at all ages responded with low-threshold potentials upon release from hyperpolarization. Slightly more than half of the LGN neurons responded to optic tract stimulation with excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), or EPSP-IPSP pairs, beginning with the youngest ages. Thus, as early as the second postnatal week, and much before the onset of pattern vision, LGN neurons have many of the membrane and synaptic properties of adult thalamic neurons. These data are consistent with LGN cells playing a significant role in activity-dependent reshaping of the retinogeniculate pathway.

The effects of pre- and posttransplantation exercise on satellite cell activation and the regeneration of skeletal muscle transplants: a morphometric and autoradiographic study in mice.

The effects of pre- and posttransplantation exercise on satellite cell activation, and the eventual regeneration of skeletal muscle transplants, were studied histologically, morphometrically and autoradiographically. It was found that satellite cells in these transplants were synthesising DNA 30 h after transplantation, and the transplants appeared to be revascularising at 60 h after surgery. Respectively, this was 18 and 12 h earlier than found in previous studies on nonexercising muscle using the same techniques. The morphometric analysis showed hypertrophy of the muscle fibres of the exercised transplants when compared with controls, and also an increase in the capillarity of the exercised transplants.

Dual activation of a sex pheromone-dependent ion channel from insect olfactory dendrites by protein kinase C activators and cyclic GMP.

Olfactory transduction is thought to take place in the outer dendritic membrane of insect olfactory receptor neurons. Here we show that the outer dendritic plasma membrane of silkmoth olfactory receptor neurons seems to be exclusively equipped with a specific ion channel activated by low concentrations of the species-specific sex pheromone component. This so-called AC1 channel has a conductance of 56 pS and is nonselectively permeable to cations. The AC1 channel can be activated from the intracellular side by protein kinase C activators such as diacylglycerol and phorbolester and by cGMP but not by Ca2+, inositol 1,4,5-triphosphate, or cAMP. Our results imply that phosphorylation of this ion channel by protein kinase C could be the crucial step in channel opening by sex pheromones.

[The effect of N-bromoacetamide on the components of sodium channel inactivation in the membrane of Ranvier's node]. / Vliianie N-bromatsetamida na komponenty inaktivatsii natrievykh kanalov membrany perekhvata Ranv'e.

In voltage clamp experiments on the frog Ranvier node, the specific protein reagent, N-bromacetamide, significantly decelerates the sodium inactivation kinetics and makes it incomplete. After treatment with N-bromacetamide, both fast and slow inactivation time constants are increased and the proportion of inactivation components is changed favouring the slowly inactivating one in the wide range of membrane potentials. The results are consistent with a single channel population following the 3-state model of inactivation.

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange.

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.

The gliding apparatus of peripheral nerve and its clinical significance.

Every nerve must have the capacity to adapt to different positions by passive movement relative to the surrounding tissue. This capacity is provided by a gliding apparatus around the nerve trunk. There is another level of gliding provided by the interfascicular epineurium which allows the fascicles to glide against one another. The clinical significance of the gliding apparatus in the context of external and internal neurolysis and nerve repair is discussed. An explanation is offered for the occurrence of the so-called meander-like deformity of fascicles, seen in nerve entrapment syndromes.

Satellite cells as blood-ganglion cell barrier in autonomic ganglia.

In a preliminary study a difference in the uptake and transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) between the trigeminal ganglion and the superior cervical ganglion was observed. After injection of WGA-HRP and HRP into the trigeminal ganglion, peroxidase was found in the space between the satellite cell processes and the ganglion cells. The ganglion cells showed pinocytosis and uptake of WGA-HRP and HRP. In the superior cervical ganglion WGA-HRP and HRP were found alongside the satellite cells but were absent in the space between satellite cells and ganglion cells. Intravenous injection revealed the presence of HRP in the space between sensory ganglion cells and their satellite cells of the trigeminal and nodose ganglion whereas HRP was absent in the space between autonomic ganglion cells and their satellite cells of the superior cervical, medial cervical and pterygopalatine ganglion although HRP lined the satellite cell membranes. By means of electron microscopy, satellite cell processes in the superior cervical ganglion were found to enwrap ganglion cells very tightly with a marginal space between both cell types. Satellite cells and their processes were mutually anchored by numerous tight junctions. In the trigeminal ganglion the extracellular space between ganglion cells and satellite cells was larger and satellite cells were found to be more loosely arranged around the ganglion cells. Satellite cell processes were only occasionally linked by tight junctions. It is concluded that satellite cells in autonomic ganglia comprise an effective barrier for WGA-HRP and HRP and probably large molecules in general. This barrier is absent in sensory ganglia.

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium.

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.

Conditional open and delay time histograms of sodium channels.

Currents through single sodium channels were recorded in neuroblastoma cells. Open time histograms were constructed from openings which appeared between 2.0 and 5.0 ms after the onset of the depolarization. Histograms constructed from openings which were not preceded by other openings showed a maximum at t greater than 0 in contrast to those, which were preceded by other openings. Time constants of delay time histograms fitted by the sum of two exponentials were different for the first, second and third records of runs. The results support the view that sodium channels have multiple open and closed states and the transition probabilities among the states depend on local conditions of the membrane.

Inhibition of reactive gliosis in vivo by exogenous axolemma and myelin fractions.

Exogenous myelin- or axolemma-enriched fractions were assessed for the ability to inhibit biochemical and morphological expressions of reactive gliosis in rat optic nerve. Elvax pellets containing exogenous myelin, axolemma, whole-brain homogenate, liver, or red cell extracts or no homogenate were inserted into a dural slit in distal regions of crushed optic nerve. Biochemical and morphological expressions of reactive gliosis were assessed at 7 or 14 days postoperatively. Post-traumatic elevations in lactic dehydrogenase activity normally seen at 7 days postoperatively were prevented by placement of Elvax pellets containing myelin or axolemmal fractions into the optic nerve. Morphological analyses indicated an inhibition of post-traumatic elevations in glial cell numbers, surface area, and nuclear size at the 14-day time point. Exposure of the axolemmal fraction to heat or trypsin inactivated its ability to modulate reactive gliotic changes. Myelin fractions were trypsin-sensitive, but not heat-sensitive. In contrast, Elvax pellets containing whole-brain tissue homogenates or liver and red cell membranes had no significant effects on post-traumatic glial changes relative to preparations in which homogenate-free pellets were used.

Solubilization of a membrane factor that stimulates levels of substance P and choline acetyltransferase in sympathetic neurons.

The choice of which neurotransmitters will be produced by a developing neuron is influenced by the microenvironment of the neuron. In this study we show that neuronal contact with membrane-associated molecules promotes expression of peptidergic and cholinergic traits. Treatment of cultured neonatal rat sympathetic neurons with plasma membranes derived from adult rat spinal cord or sympathetic ganglia induced expression of the peptide transmitter substance P and increased levels of the cholinergic biosynthetic enzyme choline acetyltransferase. The transmitter-stimulating activity could be solubilized from spinal cord membranes by the detergent octyl glucoside but not by Triton X-100. The choline acetyltransferase- and substance P-stimulating activity also could be extracted from spinal cord membranes by 4 M sodium chloride, suggesting that the active material is membrane associated rather than an intrinsic structural membrane molecule. Trypsin or heat treatment of the extract destroyed the transmitter-stimulating activity, indicating that the factor contains a protein. Activity also was destroyed by hyaluronidase treatment, suggesting that the active material may contain a glycosaminoglycan. The choline acetyltransferase-stimulating activity in the 4 M NaCl extract was eluted in a single peak from a calibrated Sephadex G-75 column with a retention time slightly less than that of a 25-kDa standard. NaDodSO4/polyacrylamide gel electrophoresis of the active peak revealed a predominant band at 29 kDa. Thus, contact-mediated stimulation of substance P and choline acetyltransferase activity in sympathetic neurons results from neuronal exposure to a 29-kDa membrane-associated factor.

Increased numbers of ion channels promoted by an intracellular second messenger.

The anomalous rectifier potassium current in Aplysia neurons was examined to determine the immediate cause of an increase in conductance induced by serotonin and mediated by adenosine 3',5'-monophosphate. Voltage-dependent cesium ion block and steady-state current power spectral density were measured under voltage clamp before and after application of serotonin. The amplitude of the anomalous rectifier conductance was increased by adding serotonin, but the shapes of the conductance-voltage curve and the power spectrum were not altered. Calculation of the number of functional channels and of the single-channel conductance from the power spectra indicates that the serotonin-induced increase in conductance resulted from an increase in the number of functional channels, while the single-channel conductance and the open-channel probability were unchanged.

Taste receptors in crayfish: recording of single nicotinamide-activated channels.

The patch clamp technique was adapted to chemoreceptive sensory cells in the claw of Austropotamobius torrentium. In cell-attached and in outside-out patches, concentrations of nicotinamide from 10(-6) mol/liter to 5 X 10(-5) mol/liter activated openings of single channels located in the cell body of the sense cell. The characteristics of these single channel openings were similar to those of neurotransmitter activated channels: the I/V curve was approximately linear, showing a reversal potential of about +15 mV and a conductance of about 33 pS. The mean open time was 1-2 ms. A similar concentration dependence was found for the activation of single channel openings as shown previously for the action potential discharge elicited by nicotinamide at the same preparation.