SYNAPSE MICROARRAY

Presented by-Preeti ChauhanVaishali KanyalBCE-III yearSYNAPSE MICROARRAY

SYNAPTOGENESISSynaptogenesis is the final stage of neural development and refers to the formation of the synapses.between neuronsbetween neurons and muscle cellsbetween neurons and glands

Synapses are the connections between neurons that allow neurons to communicate with either an electrical or chemical signal. There are about one hundred billion neurons, and each neuron has several thousand connections.Synaptogenesis begins prenatally and proceeds rapidly from birth for several yearsSynaptogenesis slows significantly later in the lifetime.Synapse elimination occurs in different areas of the brain at different times.In neurodegenerative diseases, the untimely death of neurons occur or we can say the death of neuron occurs after maturation. For example, Alzheimer the target is the distruction of synapse, hence affecting the transmission of signals.

MICROARRAYMicroarrays are small device containing microwells.The synapse microarrays consist of two main compartments connected by parallel microchannels (325 m long, 10 m wide and 3 m high) which have been previously shown to effectively isolate axons from neuronal somata Dissociated neurons are plated and cultured in compartment 1. The neural processes emanating from the somata grow through the microchannels and extend into compartment 2, which is covered by a thin (80 m) polydimethyl siloxane (PDMS) membrane held 3 m above the substrate by small posts (10 m diameter), which provide space for axonal outgrowth. The membrane also contains an array of through-holes (microwells with 30 m diameter).

SYNAPSE MICROARRAYIncreases synaptogenesis. This platform enables the induction of synaptic structures in regular arrays by precise positioning of non-neuronal cells expressing synaptic proteins, while allowing neurites to grow freely around these cells. synapse microarray technology that enables ultra-sensitive, high-throughput and quantitative screening of synaptogenesis as compared to existing technology, neuron-fibroblast coculture technique. performing a screen using traditional coculture assays involving a medium-size library of 100,000 chemicals would require several years if done with existing methods, while the time could be reduced to a few months if the screen is conducted using the synapse microarrays

For example-First, neuronal cells are loaded into well A by direct pipetting. The neurons then passively flow into compartment 1 in well B, which is coated with cell adhesion proteins. After 7 days HEK293 cells are pipetted and seeded into compartment 2 of well B as described above. Well C is used for liquid exchange. This design is also easily adaptable to 24-well, 48-well and 384-well format plates.

The HEK293 cells, express the NLG1 proteins on their membrane, which increases synaptogenesis. It has been identified that inhibitors for HDAC (histone deactetylase), are the factors responsible for the proper expression of NLG1 on the surface of HEK293 cells. For example TSA(trichostatin A).

Screening of chemical libraries using synapse microarraysIn this experiment we took the inhibitor of HDAC protein, this inhibitor increased the synaptogenesis. For example, in lab TSA is identified as this kind of inhibitor, and by using drug libraries, certain more inhibitors to HDAC were identified and were assayed using microarrays. Hence providing us the better drugs to increase synaptogenesis. Some of the drugs so identified were SAHA, LBH-589 etc and also the CN(i) series of drugs, where i represents numbers, 1-15, having difference of methylene groups only. The best drug so identified is CN13, but its presence in biological systems is under evaluations till now.

ULTRA-SENSITIVE- A doseresponse assay to measure the effect of TSA on NLG1-induced presynaptic specialization, and we compared these results to those produced by traditional coculture assays . Although traditional coculture assays allow detection of TSA-enhanced presynaptic clustering at 300 nM ,it fails to detect any effect at lower concentrations (that is, for 30 nM and 100 nM). On the other hand, the data acquired using our synapse microarray consistently showed a linear doseresponse to TSA, and we were able to detect a significant increase in NLG1-induced presynaptic clustering, even at the lowest TSA dose tested (30 nM), indicating almost tenfold improvement in sensitivity over traditional coculture assays.

SCREENING- The synapse microarray, however, is exceptionally conducive to automated imaging and analysis. We use a standard fluorescence microscope equipped with an automated stage to scan and acquire images from three fluorescent channels (corresponding to HA-NLG1, synapsin and III-tubulin). Composite images are then analysed using custom-made software that identified microwells filled with HEK293 cells, and recorded the fluorescence intensity profiles of the different channels for statistical analysis . Thus, our synapse microarrays enable full automation of both data acquisition and image analysis without any manual intervention or special equipment. Such speed-up is crucial for conducting large-scale screens.

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