2. SYNAPTOGENESIS
Synaptogenesis is the final stage of neural
development and refers to the formation of the
synapses.
1. between neurons
2. between neurons and muscle cells
3. between neurons and glands
3. 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 years
Synaptogenesis 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.
4. MICROARRAY
Microarrays are small device containing microwells.
The synapse microarrays consist of two main compartments
connected by parallel microchannels (325 μm long, 10 μm wide
and 3 μm 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 μm)
polydimethyl siloxane (PDMS) membrane held 3 μm above the
substrate by small posts (10 μm diameter), which provide space
for axonal outgrowth. The membrane also contains an array of
through-holes (microwells with 30 μm diameter).
5. SYNAPSE MICROARRAY
Increases 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
6. 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).
7. Screening of chemical libraries
using synapse microarrays
In 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
8. ULTRA-SENSITIVE- A dose–
response 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
dose–response 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
9. 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 βIII-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.
