This document discusses using neuronal networks derived from human induced pluripotent stem cells to study neurological disorders like schizophrenia. It proposes culturing the neurons under different conditions: alone, with astrocytes, or with astrocyte-conditioned media. Multielectrode arrays would record the electrical activity of the networks. Specifically, networks overexpressing the schizophrenia gene NOS1AP could be studied. The document also compares two neuronal culturing media, NbActive4 and NDM, finding that NbActive4 increases spontaneous spike rates and bursting, suggesting it improves neuronal network growth and activity.

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Branching Out: Using an In Vitro Approach to Determine
Optimal Conditions of Neuronal Activity
N. Hecht1, K. Hernandez2, and B. Firestein3
Department of Cell Biology and Neuroscience
Rutgers, The State University of New Jersey
Introduction
Accurately developed dendrite morphology is
essential for normal brain function. The
consequence of poor neuronal development is
the inability to properly receive signals from
axons (Carrel et al., 2009). Abnormal dendritic
arborization patterns have been implicated in
various neurodevelopmental disorders such as
schizophrenia. Nitric oxide synthase (NOS1AP),
a protein encoded by a schizophrenia
susceptibility gene, is an intracellular factor that
alters both neuronal morphology and synaptic
structures. The long isoform of NOS1AP
(NOS1AP-L) decreases dendrite number when
overexpressed and alters synaptic structures.
Progress has been made in understanding the
mechanisms regulating dendritic branching—the
overexpression of NOS1AP-L radically reduces
amount of dendritic branching (Carrel et al.,
2009) which influences neuronal signaling in the
brain. Therefore, culturing neurons with an
overexpression of NOS1AP might yield a
decreased burst rate, suggesting weaker network
organization.
Recent in vitro studies suggest that astrocytes
enhance synapse formation and neuronal
activity (Kucukdereli et al., 2011). It is possible
to culture human induced pluripotent stem
cell derived nueral stem cells in three
conditions: 1. Using human neurons alone, 2.
Growing neurons directly with astrocytes, 3.
Human neurons that have been cultured with
astrocyte conditioned media. The next step is
to determine the optimal combination for
hIPSCs to thrive and record electrical activity.
Once optimal conditions are met, the Firestein
lab can record activity of neuronal networks
with an overexpression of NOS1AP-L. Further
research on this in vitro neurological model
could potentially eliminate drug compounds
that induce cytotoxic effects early in the drug
discovery process. In addition, this system
would be useful to pharmaceutical companies
interested in developing new treatments, as
well as neurodevelopmental and neurological
disorder research foundations. Current animal
models used in the drug discovery process are
not sufficient to candidate drugs as they may
or may not react in the same way as human
neurons would respond. Such a reliable
neuronal network model will be useful for
screenings for novel drug therapies to treat
various neurological disorders such as
schizophrenia.
Discussion
v Neuronal network activity can be simulated
and measured using MEA technology.
v A network of human induced pluripotent stem
cell-derived neurons (hiPSC) that generates
electrical activity represents basic brain activity
and serves as an in vitro model of the human
neuronal network (Fuchs et al., 2007).
v As many neurodevelopmental disorders are
categorized defects in dendritogenesis, the
generation of recordable network activity will
allow for the use of human neurons in studies
investigating proper neuronal communication
v By using MEAs, spike rate and bursting activity
of neurons can be measured. Spike rate
corresponds to electrical activity on a single
neuron level, whereas bursting indicates the
formation of neuronal networks. Using the
non-invasive MEA technology, the action
potentials of diseased neurons (those
containing the schizophrenia susceptibility
gene, NOS1AP) can be compared to the activity
of healthy neurons. Consequently, the
neuronal network organization of neurons from
patients with neurological disorders can be
investigated.
Methods
Effect of Nueronal Culturing Media
Results
The Multielectrode Array (MEA)
MEAs, continued
v Difference between conditioning neuronal
NbActive4 and NDM is distinguishable after
measuring spike rate and bursting activity.
v Neuron networks cultured in NbActive4
v More spontaneous spike rates than
networks cultured in NDM.
v Higher number of synapses.
v Advantages include:
v Improvements in growth and viability,
and increased electrical activity in
neuronal networks
Brewer G.J., Boehler M.D., Jones T.T., Wheeler B.C.
NbActiv4 medium improvement to
Neurobasal/B27 increases neuron synapse
densities and network spike rates on
multielectrode arrays. (2008) Journal of
Neuroscience Methods, 170 (2), pp. 181-187. 4
Carrel D, Du Y, Komlos D, Hadzimichalis NM, Kwon M,
Wang B, Brzustowicz LM, Firestein BL (2009)
NOS1AP regulates dendrite patterning of
hippocampal neurons through carboxypeptidase
E-mediated pathway. J Neurosci. 29:8248-8258
Fuchs, et al. Coemergence of regulatiry and complexity
during neural network development. Dev.
Neurobiol. 67:1802-1814, 2007.
Kucukdereli H., Allen N. J., Lee A. T., Feng A., Ozlu M.
I., Conatser L. M., Chakraborty C., Workman G
Weaver M., Sage E. H., Barres B. A., Eroglu C.
(2011). Control of excitatory CNS synaptogenesis b
astrocyte-secreted proteins Hevin and SPARC.
Proc. Natl. Acad. Sci. U.S.A. 108, E440–E449.
doi: 10.1073/pnas. 1104977108.
Figure 1.
A). Standard microelectrode array layout grid
B). Schematic of MEA produced by Multichannel Systems illustrating
the location of the recording chamber surrounded by a planar
electrode array.
v Studies suggest NbActive4 is an improved
neuronal culturing media
v Optimized to increase spontaneous spike
rates for rat embryonic hippocampal
neurons.
v NbActive4 includes
v Nuerobasal neuronal culturing media
v Used as the standard culturing media
since 1993
v Supplements of reatine, cholesterol, and
estrogen.
v Studies report an 8-fold increase in spontaneous
spike activity which suggests that NbActive4 is an
improvement to Nuerobasal/B27 for neuronal
networks (Brewer et al., 2008).
v The electrical activity of neuronal networks
can examined over several weeks or months
in vitro so long as the cultures are
conditioned in appropriate serum-free
media
v Neurons are cultured in NbActive4
media and NDM.
v Series of electrodes on MEA detect changes
in voltage from the isolated region
v Activity of human nueronal cultures
measured at 37oC and converted into
signals by data acquistion software –
MCRack by MultiChannelSystems
v Spike rate and bursting activity
measured and compared for the
human neurons conditioned in
the two different neuronal
culturing medias, NDM and
NbActive4.
A. B.
MCRack
Figure 2.
A). Representative spike (A) and spontaneous bursting events (B).
Spikes indicate that the signals surpassed a set threshold level
whereas bursting events indicate periods of high activity.
Figure 3.
NbActive4 increases spontaneous spike rates and bursting compared
cultured in Neurobasal/B27. Activity (µV) is compared over the
indicated times.4
References