Bruce G. Wallace

Inhibition of agrin-induced acetylcholine-receptor aggregation by heparin, heparan sulfate, and other polyanions

Heparin and heparan sulfate have been shown to block nerve-induced acetylcholine-receptor (AChR) aggregation at developing neuromuscular junctions. We found that heparin, heparan sulfate, and a wide variety of other polyanions also inhibited agrin-induced AChR aggregation. The more highly charged the polyanion, the more potent it was as an inhibitor. Inhibition of agrin-induced AChR aggregation was due, at least in part, to the formation of a complex between the polyanion and agrin that was inactive. These findings are consistent with the hypothesis that nerve-induced aggregation of AChRs is mediated by the release of agrin, or a closely related protein, from axon terminals and suggest that a polyanion, such as a sulfated proteoglycan, may be involved in the interaction of agrin with its receptor on the myotube surface.

Molecular Components of the Synaptic Basal Lamina That Direct Differentiation of Regenerating Neuromuscular Junctions

Results of experiments outlined here provide evidence that components of the myofiber basal lamina sheath direct the formation of active zones in regenerating motor nerve terminals and the development of infoldings and the aggregation of AChRs in the plasma membrane of regenerating myofibers. As a step toward identifying the basal lamina molecules that aggregate AChRs, we are now studying an ECM fraction from the Torpedo electric organ that causes AChRs to aggregate on cultured myotubes. We have solubilized and purified the electric organ AChR-aggregating molecules over 1000-fold. Only nanogram amounts of the most purified extracts are required to cause detectable AChR aggregation. We have also shown that similar activity can be extracted in relatively small amounts from muscle. Antiserum raised against the partially purified electric organ material completely blocked and immunoprecipitated the AChR-aggregating activity in extracts of the electric organ and muscle and bound to components of the basal lamina of frog muscle fibers. Although several polypeptides are present in our most purified extracts, an antiserum against polypeptides in the range of 80 kD completely blocked AChR aggregation by soluble extracts of the electric organ. These findings demonstrate the feasibility of isolating molecules from the synapse-rich electric organ that cause AChR aggregation and comparing them by immunological techniques with those in basal lamina at the neuromuscular junction.

Regeneration of synaptic connections by sensory neurons in leech ganglia maintained in culture.

Leech ganglia maintained in organ culture were used to follow regeneration and the formation of synaptic connections by individual neurons. From earlier physiological studies on operated animals it is known that the c. n. s. of the leech is able to regenerate and that specific connections can be reformed. The present experiments show that specific regeneration also occurs in vitro but that axons do not simply grow directly back to their targets. (1) Axons were severed by crushing the connectives linking pairs of ganglia at the time of removal from the animal. Light and electron microscopy indicated that the procedure of crushing severed all the axons within the connectives. For several days after the lesion had been made, conduction of impulses from one ganglion to the next was abolished. (2) After 5–10 days in culture, stimulation of the connectives with external electrodes gave rise to impulses that were once again conducted beyond the site of the lesion. Characteristic excitatory and inhibitory synaptic potentials were evoked in identified sensory and motor cells in both ganglia by this indiscriminate stimulation of axons. Electron micrographs of the crushed region showed not only regenerated axons traversing the site of the lesion but also synaptic profiles similar to those seen in the neuropile of normal ganglia. Thus, pre- and post-synaptic specializations had been formed during regeneration in a part of the c. n. s. where they are not normally present. (3) Individual sensory neurons were injected with horseradish peroxidase to reveal the course taken by their regenerating axons. At the site of the crush profuse branching occurred by 7 days. The arborization of a single axon was highly complex, with many varicosities present on fine branches. After two weeks in culture, one or more of the processes had usually grown beyond the crush and in certain instances had reached the next ganglion. Other branches ran back towards the ganglion in which the cell body was situated. During the period of the experiments (up to 45 days) no retraction of the sprouted fibres or of the arborization at the crush was observed. In addition to sprouting at the site of the lesion considerable sprouting also occurred within the ganglion, close to the cell body. (4) Individual mechanosensory neurons regenerated and would once again evoke synaptic potentials in their original targets after two weeks in culture. Thus, intracellular stimulation of single sensory cells in one ganglion gave rise to synaptic potentials in the appropriate motor neuron of the neighbouring ganglion. Injection of such sensory cells with horseradish peroxidase showed that their axons had extended beyond the lesion and ramified in the neuropile of the next ganglion. (5) It is concluded that neurons in leech ganglia are able to regenerate and reform appropriate synaptic connections in culture. The degree of precision is hard to assess because of novel synaptic interactions and numerous additional sprouts that develop during regeneration.

Distribution of AChE in cholinergic and non-cholinergic neurons

Intracellular and surface acetylcholinesterase activities were determined for individual cholinergic and non-cholinergic neurons dissected from the central nervous system of the leech. Echothiophate pretreatment was used to inhibit selectively extracellular enzyme. Cells releasing acetylcholine as a transmitters had approximately 10-fold higher levels of intracellular acetylcholinesterase activity, while all neurons had similar levels of activity associated with the cell surface. These results suggest that intracellular cholinesterase may be a useful marker for cholinergic neurons.

Chapter 32 Agrin

Publisher Summary This chapter reviews studies on agrin, which is likely to be similar to the extracellular synaptic organizing molecules (ESOMs) that cause acetylcholine receptors (AChR) and acetylcholinesterase (AChE) aggregation at the regenerating neuromuscular junction. Monoclonal antibodies against agrin recognize molecules highly concentrated in the neuromuscular junctions synaptic cleft. Agrin causes a 3- to 20-fold increase in the number of AChR aggregates on cultured chick myotubes without influencing myotube size. There is no discernible increase in total AChR number on the myotube surface, nor is there a change in the AChR degradation rate. The AChR aggregating effect is dose-dependent and is because, at least in part, of lateral migration of AChRs present in the muscle cell plasma membrane at the time agrin is applied. Extracts containing agrin cause the formation of AChE and butyrylcholinesterase (BuChE) aggregates on cultured chick myotubes. Monoclonal antibodies against at least five different agrin epitopes recognize molecules at the Torpedo neuromuscular junction. Three antibodies against agrin stain the neuromuscular junction in the muscles of other species.

Square Kilometre Array: The radio telescope of the XXI century

The Square Kilometre Array (SKA) will be the world’s largest and most sensitive radio telescope. It will address fundamental unanswered questions about our Universe including how the first stars and galaxies formed after the Big Bang, how dark energy is accelerating the expansion of theUniverse, the role of magnetism in the cosmos, the nature of gravity, and the search for life beyond Earth. This project envisages the construction of 133 15-m antennas in South Africa and 131072 log-periodic antennas in Australia, together with the associated infrastructure in the two desert sites. In addition, the SKA is an exemplar Big Data project, with data rates of over 10 Tbps being transported from the telescope to HPC/HTC facilities.

Fiber-to-the-telescope: MeerKAT, the South African precursor to Square Kilometre Telescope Array

Abstract. Scientific curiosity to probe the nature of the universe is pushing the boundaries of big data transport and computing for radio telescopes. MeerKAT, the South African precursor to Square Kilometre Array, has 64 antennas separated by up to 12 km. By 2018, each antenna will stream up to 160 Gbps over optical fiber to a central computing engine. The antenna digitizers require highly accurate clock signals distributed with high stability. This paper outlines requirements and key design aspects of the MeerKAT network with timing reference overlay. Fieldwork results are presented into the impact of birefringence and polarization fluctuations on clock stability.

Robust fiber-based frequency synchronization system immune to strong temperature fluctuation

In order to make the fiber-based frequency synchronization system suitable for the use of large-scale scientific and engineering projects in which the ambient temperature of the fiber links change dramatically, we design a non-harmonic frequency dissemination system immune to strong temperature fluctuation. After the lab tests, in which the ambient temperature of the fiber fluctuates 40°C/day and 20°C/h, respectively, the relative frequency stabilities of this system reaches 4.0 × 10−14/s and 3.0 × 10−16/104  s. It is demonstrated that the proposed non-harmonic scheme shows a strong robustness to complicated working environment with strong temperature fluctuation.