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Current Stem Cell Research & Therapy, 2010, 5, 251-260
251
Role of Astrocytes, Soluble Factors, Cells Adhesion Molecules and Neurotrophins in
Functional Synapse Formation: Implications for Human Embryonic Stem Cell
Derived Neurons
Mahesh C. Dodla, Jennifer Mumaw and Steven L. Stice*
Regenerative Bioscience Center, University of Georgia, Athens, GA, USA
Abstract: Availability of human embryonic stem cells (hESCs) and its neural derivatives has opened up wide possibilities of using these
cells as tools for developmental studies, drug screening and cell therapies for treating neurodegenerative diseases. However, for hESCderived neurons to fulfill their potential they need to form functional synapses and spontaneously active neural networks. Until recently
very few studies have reported hESC-derived neurons capable of forming such networks, suggesting lack of certain components in culture media to promote mature synaptogenesis. In this review we discuss the various factors that enhance functional synapse formation in
primary and stem cell-derived neuronal cultures. These factors include astrocytes, astrocyte-derived factors, cell adhesion molecules and
neurotrophins. We discuss the current literature on studies that have used these factors for functional differentiation of primary neural
cultures, and discuss its implications for stem cell -derived neural cultures.
Keywords: Human embryonic stem cells, neural differentiation, functional synapse, glial cells, neurotrophins, cell adhesion molecules.
1. INTRODUCTION
Human embryonic stem cells (hESCs) are pluripotent cells
which possess multiple defining characteristics that make them
attractive as research tools, such as the ability to maintain unlimited
proliferation while retaining the ability to differentiate in vitro and
in vivo into cell types of all three germ layers [1]. In addition,
hESCs are also amenable to directed differentiation. Using ectodermal differentiation protocols, an unlimited supply of neural progenitor cells and neurons can be derived from hESCs [2, 3].
Moreover, differentiation protocols have been tailored to allow the
selective derivation of specific neuronal sub-types [4-6]. Directed
neurogenesis from hESCs provides opportunities for generating
cells for transplantation therapies to treat neurogenerative diseases,
study pharmaceutical effects on neural progenitors and differentiated neurons, and study neurogenesis during early embryonic development [7-11]. However, to fulfill these promises hESC-derived
neurons first need to be able to develop into functional neuronal
networks in vitro, and integrate into existing neural networks after
in vivo transplantation. These goals which have been difficult, if not
elusive, to achieve are essential to realize the full potential of
hESC-derived neurons.
Neural networks can be described as populations of synaptically
interconnected neurons capable of generating electrophysiological
activities which spread both spatially and temporally, thus mimicking the basic principle of brain activity. Simultaneous monitoring of
a large population of neurons has given us an understanding of how
neuronal networks in brain process information. Previously, such
studies used neurons from non-human sources due to the difficulty
in availability of human neural tissue. However, hESC-derived
neural stem cells now provide an unlimited source of neural cells
for similar studies, and are a better indicator of human response.
One caveat to using hESC-derived neural cells is the lack of functional network formation in basal culture conditions. For hESCderived neurons to form functional networks, they need to be functionally active (i.e. have active membrane properties) and also form
functional synapses with other cells. hESC-derived neurons with
active membrane properties have been previously described on the
single cell level in vitro using the patch clamp technique [12-14].
*Address correspondence to this author at the Regenerative Bioscience
Center, University of Georgia, 425 River Rd., Athens, GA 30602, USA;
Tel: 706-583-0071; Fax: 706-542-7925; E-mail: [email protected]
1574-888X/10 $55.00+.00
However, very little data has been published indicating hESC- derived neuron’s ability to form functional synapses and neural networks that are spontaneously active. Until recently only a couple of
publications have demonstrated the generation of spontaneously
active neural networks through directed differentiation of hESC
[13, 15]. This suggests that more work needs to be done to optimize
the generation of functional neural networks from hESC-derived
neurons. Co-culture with rat primary astrocytes has been shown to
hasten functional network formation in hESC-derived neurons [13,
16]. However, addition of xenogenous cell types and external
stimulation are not ideally suited for applications such as drug
screening studies or cell therapy. Development of spontaneously
active neural networks, without such external aid, will be essential
for hESC-derived neurons to fulfill their promise for developmental
and drug screening studies, as well as for regenerative medicine.
Most of our understanding of neuronal synapse formation
comes from studies on primary neuronal cultures, and very little is
known about functional synapse formation in mESC- and hESCderived neuronal cultures. In this article we review the contribution
of glial cells, glial and neuronal derived factors and cell adhesion
molecules towards functional synapse formation in primary neuronal cultures. We then discuss how these can be applied to derive
functionally active hESC-derived neural networks.
2. ARCHITECTURE OF SYNAPSES IN CENTRAL NERVOUS SYSTEM
Communication between neurons and their target organs/ cells
occurs through specialized sites of cell-cell contact called synapses.
Synapses between neurons in the central nervous system (CNS) are
called as CNS or central synapses, while those between motor neurons and muscle fibers are termed as neuro-muscular junctions
(NMJs). In this review, we will restrict our discussion to central
synapses as they are structurally similar to synapses in ESCsderived neuronal cultures in vitro, and have similar mechanisms for
synaptogenesis.
Although there are many different kinds of synapses in the
CNS, all synapses can be divided into two general classes: electrical
synapses and chemical synapses. Electrical synapses permit direct,
passive flow of electrical current from one neuron to another
through gap junctions, which are specialized membrane channels
that connect the two cells and allow bidirectional flow of current. In
contrast, at chemical synapses the pre-synaptic cell is the sender of
the signal and the post-synaptic cell is the recipient of the signal.
Both the pre- and post-synaptic cells have complex structural com© 2010 Bentham Science Publishers Ltd.
252 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3
ponents which make them adept in their respective roles. Presynaptic cells communicate through the release of neurotransmitters
which bind to receptors on the post-synaptic cell. These neurotransmitter receptors are further classified as ionotropic or metabotropic receptors. Ionotropic receptors are a group of transmembrane ion channels that open or close in response to binding to neurotransmitters. On the other hand, upon binding of neurotransmitters to metabotropic receptors, ion channel function may be modified through the action of second messengers or other signal transduction mechanisms.
Synapses are highly asymmetric cellular junctions designed for
rapid and repetitive signaling between neurons and their targets. A
synapse comprises of 3 distinct components: a pre-synaptic specialization, a synaptic cleft, and a post-synaptic specialization (Fig.
(1)). The pre-synaptic specializations have two characteristic features. First, they contain hundreds to thousands of synaptic vesicles
(SVs) containing neurotransmitters in the form of neuropeptides,
biogenic amines, amino acids, or purines. In addition, the diffusion
of novel messengers, nitric oxide and carbon monoxide, to the postsynaptic cell are also capable of conducting signals through chemical synapses. Second, the pre-synaptic plasma membrane contains
specialized region called the active zone (AZ), where the synaptic
vesicles dock, fuse, and release their components into the synaptic
cleft. The active zone is characterized by the presence of a collection of proteins that appear, by electron microscopy, as an electron
dense matrix associated with the cytoplasmic face of the presynaptic membrane. The post-synaptic plasma membrane, which
lies opposite to the active zone, is a second electron-dense structure
and is referred to as post-synaptic density (PSD). Similar to the
active zone, the PSD is defined by a large collection of structural/scaffold proteins that serve to cluster neurotransmitter receptors, cell adhesion molecules and a diverse set of other signaling
proteins at high density. The pre-synaptic and post-synaptic components are separated by a gap called the synaptic cleft, which contains largely uncharacterized matrix of cell adhesion molecules
(CAMs) and extracellular matrix (ECM) proteins. The pre- and
Dodla et al.
post-synaptic membranes are bound to each other through the
CAMs and ECM proteins.
Classification of synapses is also done based on synaptic function, i.e., whether synapse is excitatory, inhibitory, or modulatory
and whether it activates G-protein coupled receptors (metabotrophic), or ion receptors (ionotrophic). In this review, we restrict our
comments to excitatory glutamatergic central synapses because
most of the insights into the molecular mechanisms of synaptogenesis have come from studies on these synapses.
2.1. Differentiation of Glutamatergic Pre-Synaptic Terminal in
Primary Cultures
The initial contact between any two neurons in CNS is followed
by several intermediate steps before a fully functional synapse is
established. These intermediary steps involve the differentiation of
the pre-synaptic terminal and the opposing post-synaptic terminal,
resulting in a mature synapse.
The differentiation of the pre-synaptic terminal includes the appearance of synaptic vesicle (SV) clusters and the formation of
active zones (AZs). The SVs are membrane-bound compartments
containing neurotransmitter molecules. When stimulated, the SVs
fuse with the pre-synaptic terminal at the AZ, followed by exocytosis of neurotransmitters into the synaptic cleft (Fig. (1)).
The AZ is a vital component to the correct formation and function of a synapse. In transmission electron microscopy (TEM)
analysis, AZ show up as a dense area due to increased heavy metal
staining, indicating the dense protein nature of AZ. The structure of
the AZ allows the pre-synaptic cells to be primed for and to complete transmission. AZ serves a multitude of functions. AZs facilitate the clustering, tethering, docking, and fusing of the SVs with
the cellular membrane and releasing the neurotransmitters into the
synaptic cleft.
The pre-synaptic nerve terminal contains voltage-gated calcium
channels which allow calcium to enter in response to action potentials in the pre-synaptic cell. The entry of calcium into the pre-
Fig. (1). The synapse consists of 3 distinct components: pre-synaptic terminal (A), synaptic cleft (B) and the post-synaptic terminal (C). At the pre-synaptic
terminal (A), the synaptic vesicles uptake neurotransmitters and then release them into the synaptic cleft. The scaffolding proteins: piccolo and bassoon, mediate
docking of synaptic vesicles, while the SNARE proteins: synaptobrevin, SNAP-25 and syntaxin mediate synaptic vesicle priming. Synaptotagmin prevents
synaptic vesicle fusion until it is activated by calcium ions, upon which the vesicles fuse with cell membrane and release neurotransmitters in to the synaptic
cleft (B). The post-synaptic terminal (C) contains numerous receptors, such as, NMDAR, AMPAR and mGluR, which are activated by glutamate and mediate
post-synaptic signaling. Several cell adhesion molecules, such as neuroligin- neurexin, also help in synapse formation and maturation.
Role of Astrocytes, Soluble Factors, Cells Adhesion Molecules
synaptic cell induces SVs to fuse with the cell membrane within 0.2
ms. At the AZ, special proteins called SNAREs (Souble Nethylmaleimaide sensitive factor attachment protein receptors)
tether the SVs in close proximity to the synapse and mediate the
fusion of SVs to membrane upon calcium activation. There are two
types of SNARE proteins, the v-SNAREs and the t-SNAREs. The
v-SNAREs are vesicle associated SNAREs such as synaptobrevin2
(also known as Vesicle associated membrane protein 2, VAMP2),
while the t-SNAREs are target membrane associated SNAREs such
as syntaxin1 and SNAP-25 [17]. The v-snares associate with and
mediate the “docking” of the SVs near the synaptic membrane. To
be “primed” for neurotransmitter release the v-SNAREs and the tSNAREs interact, bringing SVs close to the cell membrane, shown
in Fig. (1). Synaptotagmin, another synaptic vesicle protein, acts as
a clamp and inhibits vesicle fusion [17]. Without calcium stimulation the SNAREs maintain an inhibitory role which prevents erroneous transmission. Once calcium enters the cells, it binds to synaptotagmin and prevents its inhibitory function, this results in the vSNAREs and the t-SNAREs altering conformation. This alteration
in conformation results in fusion of the SVs with the cell membrane
and release of neurotransmitters into the synaptic cleft (Fig. (1)).
Apart from the SNARE complex proteins the AZ is characterized by the presence of proteins such as Piccolo, Bassoon, and Rab3
interacting molecule (RIM), which have been proposed to provide a
scaffold for the localization of synaptic proteins, and help to define
the site of SV docking, fusion and neurotransmitter release, as
shown in Fig. (1) [18-20]. In addition, cell adhesion molecules
(CAM) are present at the pre- and post-synaptic terminals and act as
“priming” and “inducing” molecules for synapse formation. Some
of the CAMs at the pre-synaptic terminal are: neurexins, cadherins
and integrins. Through physical interactions, these proteins form
scaffolds at the AZ and play a role in organizing the release and
retrieval of SVs, and in regulating changes in release during shortterm and long-term form of synaptic plasticity.
2.2. Differentiation of Glutamatergic Post-Synaptic Terminal in
Primary Cultures
The membrane specialization of the post-synaptic cell that
serves to receive information sent by the pre-synaptic cell is called
as post-synaptic specialization or post-synaptic density (PSD). The
PSD also appears as an electron-dense area in TEM analysis due to
the accumulation of proteins at the region of post-synaptic membrane, opposite to the AZ. It is now believed that there are several
hundreds of proteins present in the PSD structure. The PSD comprises of large multimolecular complexes that are composed of
dozens of molecules: receptors, scaffolds and adaptor molecules,
cell adhesion molecules (CAMs), cytoskeleton proteins, protein
kinases, protein phosphatases and other signaling molecules.
Glutamate receptors are the main components of and are highly
concentrated in the excitatory PSD. The major post-synaptic glutamate receptors include N-methyl-D-aspartate (NMDA) receptors, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors, and the group I metabotropic glutamate receptors
(mGluRs). The targeting of AMPA- and NMDA-type glutamate
receptors to synapses in the central nervous system is essential for
efficient excitatory synaptic transmission (Fig. (1)). However, the
NMDA receptors are a consistent feature of excitatory synapses of
the forebrain, whereas AMPA receptor content is highly variable;
indeed, a significant fraction of excitatory synapses lack AMPA
receptors altogether.
NMDA receptors are hetero-tetrameric structures composed of
NR1 and NR2 subunits which possess long cytoplasmic tails. The
C-terminal of the long cytoplasmic tails contains conserved sequence –ESDV or -ESEV, which bind to the PDZ domains of synapse associated protein-90 (SAP90)/PSD-95, an abundant constituent of the PSD. The PSD-95/SAP90 belong to the membrane associated guanylate kinase (MAGUK) superfamily of proteins, which
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are characterized by the presence of PDZ domains, a Src homology
3 domain, and a guanylate kinase-like (GK) domain. The interaction between NR2 subunits of the NMDA receptor and PSD-95 is
important for the specific localization of receptors in the PSD, in
the coupling of NMDA receptors to cytoplasmic signaling pathways, and in the anchoring of NMDA receptors to the post-synaptic
cytoskeleton. In the NMDA/PSD-95 complex, the GK domain of
the PSD-95 binds to an abundant family of proteins in the PSD,
termed guanylate kinase associated protein (GKAP) which in turn
binds to Shank, a family of scaffold proteins. Shank interacts with
Homer, which was originally identified as a binding partner for
group I mGluRs. The NMDA receptor/PSD-95 complex therefore,
via Shank and Homer, is potentially linked to mGluRs (Fig. (1)).
Shank and Homer may also contribute to functional coupling between NMDA receptors and intracellular calcium stores, cytoplasmic proteins, smooth endoplasmic reticulum, and the post-synaptic
cytoskeleton.
AMPA receptors, like NMDA receptors, are hetero-tetrameric
structures composed of GluR1-4 subunits. The C-terminal cytoplasmic tails of AMPA receptor subunits interact with a distinct set
of cytoplasmic proteins than do NMDA receptors. The GluR2 and
GluR3 subunits of AMPA receptors share a C-terminal sequence
(SVKI) that interacts with the PDZ domain of glutamate receptor
interacting protein (GRIP)/AMPA receptor-binding protein (ABP),
a family of proteins, and protein interacting with C kinase 1
(PICK1). Interaction between the C terminus of AMPA receptor
subunits and PDZ domain scaffold proteins appear to be important
for synaptic targeting and stabilization of AMPA receptors. The
AMPA receptor subunit also binds to N-ethylmaleimide-sensitive
fusion protein (NSF), an ATPase involved in membrane fusion and
vesicle trafficking, which regulates synaptic plasticity by regulating
vesicle trafficking or protein unfolding of AMPA receptors. In
summary, the excitatory PSD is therefore a complex network of
proteins that supports receptor’s post-synaptic actions and modulates the receptor’s activity.
3. GLIAL CELLS AND SYNAPSE FORMATION IN PRIMARY AND ESC-DERIVED NEURONAL CULTURES
3.1. Introduction
Glial cells represent the most abundant cell population in the
central nervous system (CNS). There are 3 types of glial cells in the
CNS: astrocytes, oligodendrocytes and microglia. Astrocytes are
the most abundant glial cells with irregular star-shaped cell bodies
and broad end-feet on their processes. They interact extensively
with neurons and provide structural and metabolic support. Oligodendrocytes are smaller than astrocytes, and have spherical or
polyhedral cell bodies with a small amount of cytoplasm around the
nucleus. Oligodendrocytes have several slender processes which
wrap around axons, forming myelin sheaths. Microglia, the smallest
of the glial cells, act as phagocytes, the immune cells of the CNS, to
clean up debris [21]. Previously glial cells (‘glia’ originates from
the Greek word for glue) were thought to be responsible only for
providing structural support to neurons and providing them with
trophic factors essential for their survival, while the function of
transmitting and processing information was attributed exclusively
to neurons. This concept has changed drastically in the last few
years. It is now widely believed that the glial cells play a major role
in brain development and function [22-25]. Glia and neurons which
are intertwined throughout the nervous system are functionally
interacting, and the key site of interaction is the synapse. In the next
few sections we discuss some of the factors affecting synapse formation, also summarized in Table 1.
3.2. Role of Glial Cells in Formation of Synapses in Primary
Cultures
Throughout the nervous system, the glial cells are closely associated with synapses. Studies with primary cultures of rat cortical
254 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3
Table 1.
Dodla et al.
Summary of Factors Enhancing Synapse Formation
Factors Enhancing Synapse Formation
Effects on Synapse Formation (References)
1. Addition of glial cells
Increases expression of synaptophysin, GABAA receptor, NMDA-R1 receptor, Glu-R1 receptor and enhances
spike rates [26-28].
2. Cholesterol
Enhances the efficacy of presynaptic transmitter release, localization of pre- and post-synaptic puncta, frequency of
spontaneous events [30,35,36].
3. Estradiol
Increases spine synapses and synaptic proteins, increases pre-synaptic puncta, spontaneous synaptic current and
vesicle release [40,41].
4. Thrombospondins
Increase pre-synaptic puncta and vesicle recycling, but do not increase synaptic transmission [33].
5. Neurotrophins (BDNF, NGF, NT-3/4/5)
Increase dendritic and axonal branching, increase number of SVs, localization of SVs at active zone, number and
localization of post-synaptic NMDA receptor, GABAA receptor and mGluR [56-65].
6. Cell adhesion molecules and diffusible factors
• Narp, EpherinB- EphB
• Increase clustering of AMAP and NMDA receptors, dendritic spine development and maturation [84-89].
• SynCAM, Neuroligins and neurexins
• Induce synapse specification, adhesion and signaling; induce pre-synaptic and post-synaptic differentiation [73,
90-92].
AMPA: -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
BDNF: Brain derived neurotrophic factor
GABA: Gamma aminobutyric acid
Narp: Neuronal activity-regulated pentraxin
NMDA: N-methyl-D-aspartate
NGF: Nerve growth factor
SV: Synaptic vesicle
SynCAM: Synaptic cell adhesion molecule
neurons have shown that glial cells significantly promote synaptogenesis [26, 27]. When purified retinal ganglion cells (RGCs) were
cultured at 99.5% purity, devoid of glial cells, only a few synapses
were established. The synapses displayed normal ultrastructure
morphology but little spontaneous electrical activity, and high failure rate [27]. On the other hand, in co-cultures with neuro-glia, few
failures were detected and the frequency and amplitude of spontaneous synaptic events were both strongly enhanced. The efficacy of
evoked synaptic transmission was drastically enhanced as well.
Similar results were observed with networks of rat hippocampal
neurons cultured in serum-free conditions, with and without addition of astroglia, on multi-electrode arrays [28]. Addition of astroglia to the hippocampal neuronal networks enhanced spontaneous
spike rates, as well as glutamate-stimulated spiking. Addition of
astroglia also leads to increased expression of synaptophysin (a SV
associated protein), GABAA receptor, NMDA-R1 receptor, and
Glu-R1 receptor. Together, these results indicate that addition of the
astroglia increased the density of synapses and receptors, and facilitated higher spike rates in the network [28].
3.3. Glial-Derived Factors in Formation of Synapses in Primary
Cultures
The effects of glial cells on neuronal synapse formation could
be mimicked when astrocytic- or oligodendrocytic-conditioned
media was added to the glial-free neuronal cultures, suggesting that
a diffusible factor(s) released by glial cells was acting on neurons
and promoting synapse formation. The factors suspected to induce
synapse formation and synaptic plasticity include glutamate [29],
cholesterol [30], TNF- [31], ATP [32], thrombospondins [33], and
glial protein S100b [34].
3.3.1. Role of Glial-Derived Cholesterol in Formation of Synapses
in Primary Cultures
Retinal ganglion cells (RGCs) form significantly more synapses
when cultured in the presence of astrocytes than cultures without
astrocytes in serum-free media [27]. Treatment of glia-free microcultures of RGCs with glia-conditioned medium (GCM) increased
the number of synapses per neuron by up to 10-fold, as compared to
serum-free media cultures [35]. There was a similar increase in the
frequency of spontaneous events, the number of FM1-43 dyelabeled functional release sites, and of puncta, where pre- and postsynaptic markers co-localized. In addition, GCM treatment en-
hanced the efficacy of pre-synaptic transmitter release as indicated
by lower failure rates of stimulation-induced excitatory synaptic
currents, a 200-fold increase in the frequency of asynchronous release and an accelerated stimulation-induced FM1-43 destaining.
The studies summarized above show that soluble factors in
GCM strongly promote synapse development. Analysis of GCM
fractions and their effects on synaptogenesis identified a diffusible
astrocyte-derived factor, cholesterol, in a complex with apolipoprotein E-containing lipoproteins, as a limiting factor critical for neuronal synapse formation [30, 36]. Role of cholesterol in synaptogenesis was confirmed by several assays. First, reduction of cholesterol concentration in GCM by an inhibitor of cholesterol synthesis,
mevastatin, strongly reduced its synaptogenic effect. Second, the
effect of GCM on synaptic activity was reduced by blocking lipoprotein-bound cholesterol uptake by a competitive low-density
lipoprotein (LDL)-receptor antagonist. Third, GCM strongly raises
the cholesterol content of RGCs as shown by the cytochemical
staining of RGCs with a cholesterol-binding drug. Finally, addition
of cholesterol alone mimicked most of the GCM-induced effects on
synapse number and efficacy [35]. Other similar studies suggest
that the ability of CNS neurons to form synapses is limited by the
availability of cholesterol [30]. In pure cultures of RGCs, the neurons produce enough cholesterol to survive, grow axons and dendrites and form a few immature synapses. However, the formation
of numerous, efficient, and mature synapses required additional
amounts of cholesterol from glial cells. Thus, the availability of
cholesterol appears to limit synapse development. This may explain
why most synapses in the developing brain are formed after the
differentiation of glial cells [37, 38], and neurobehavioral manifestations result from defects in cholesterol or lipoprotein homeostasis
[39].
3.3.2. Role of Glial-Derived Estrogen in Formation of Synapses in
Primary Cultures
Studies in rodent neural cultures have suggested that cholesterol-promoted synapse formation results from the function of cholesterol acting as precursor of estradiol (predominant form of estrogen) synthesis in some parts of the brain [40]. Hippocampal cultures treated with cholesterol showed an increase in estradiol release into the medium, an increase in the number of spine synapses
and immunoreactivity of synaptic proteins [40]. However, blockade
Role of Astrocytes, Soluble Factors, Cells Adhesion Molecules
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255
of cholesterol to estrogen conversion using letrozole, a potent aromatase inhibitor, or blockade of estrogen receptors lead to abolition
of cholesterol-induced synapse formation. Further, a knock-down of
steroidogenic acute regulatory protein was developed, in which the
access of cholesterol to the first enzyme of steroidogenesis was
blocked. A rescue of the reduced synaptic protein expression in the
transfected cells was achieved by estradiol and not by cholesterol.
This suggests that conversion of cholesterol to estradiol was required for synapse formation in hippocampus.
Role of estradiol in synapse formation was also observed in
neonatal rat cortical neurons [41]. The concentration of estradiol in
cortical astrocyte conditioned media (ACM) increased with time
and peaked around day 14 of culture. Addition of ACM to pure
cortical neuronal cultures increased the number of pre-synaptic
puncta by nearly six-fold, and enhanced the incidence and mean
amplitude of spontaneous synaptic current [41]. At the same time, it
also altered kinetics of vesicle release. Addition of tamoxifen (estrogen receptor antagonist) to the culture blocked the ACMmediated increase in synaptic formation and transmission, suggesting a role for estrogen. Further, addition of estradiol to the culture
media mimicked the effect of ACM on synaptic formation and
transmission. This suggests that astrocyte-derived estradiol plays an
important role in the formation and activity of synapses.
3.3.3. Role of Glial-Derived Thrombospondins in the Formation
of Primary Synapses
Another class of astrocyte secreted extracellular matrix
proteins, thrombospondins (TSPs)-1 and -2, also promote CNS
synaptogenesis in vitro and in vivo. TSPs are large ECM proteins
that can mediate both cell-cell and cell-matrix interactions [42].
Addition of TSPs-1 or -2 to cultured RGCs resulted in a large
increase in number of new synapses, to the same level as that induced by the presence of astrocytes [33]. TSP-1 and -2 are both
expressed in the developing brain during the peak period of synaptogenesis but shut-off by adulthood. TSP1/TSP2 double mutant
mice exhibit a dramatic 30% reduction in the number of synapses
formed during postnatal stages [33]. In contrast to the fully
functional synapses induced by astrocytes, the synapses induced by
TSPs are post-synaptically silent, i.e., although the TSPs increased
the pre-synaptic puncta and vesicle recycling, they did not increase
synaptic events as measured by whole-cell patch clamp. These
findings indicate that there are additional unknown factors secreted
by astrocytes, together with TSPs, that promote the maturation of
functional synapses.
ences or related to developmental stage specificity. Wu et al studied
the neuronal differentiation bias in two hESC lines [16]. The
hESC–derived neurons demonstrated normal electrical properties
but failed to exhibit spontaneous or evoked synaptic activity even
after several weeks. However, growing the human neurons on monolayer of astrocytes from newborn mice evoked spontaneous synaptic events.
Johnson et al. have demonstrated that without exogenous astrocytes it takes 9 weeks for hESC–derived neurons to demonstrate
significant localization of synapsin-1 puncta along with MAP2 +
dendrites [13]. Spontaneous synaptic activity in 40% of cells was
first seen at 7 weeks of culture. Interestingly, outgrowth of astrocytes within the culture was also observed at 7 weeks, suggesting
that contribution of astrocytes is essential for synaptogenesis and
synaptic transmission. Heikkilä et al. have demonstrated differentiation of hESCs into neuronal cultures with spontaneous activity [15].
After 5 weeks of differentiation from hESCs to neural aggregates,
when plated on Multi-electrode arrays (MEAs) the neural network
activity developed from single spikes to spontaneous bursts in the
time frame of 1 month on MEA dishes. The abundance of burst
activity in the cultures indicates robust functional synapse formation and network activity. Reversible addition of TTX blocked sodium channels and the network activity, demonstrating that the
network activity was dependent on voltage-gated sodium channels.
Presence of functional excitatory glutamatergic synapses and inhibitory GABAergic synapses was demonstrated using appropriate
antagonists.
From the above mentioned studies it suggests that glial differentiation of hESCs is essential for the neurons to exhibit spontaneous functional synaptic activity, or exogenous astrocytes need to be
added to induce spontaneous activity. Since glial differentiation
from hESCs takes several weeks, so does spontaneous synaptic
activity. One way to hasten synaptogeneis could be by directly adding glial-derived factors to hESC-derived neural cultures. There is
no published data on the individual effects of cholesterol, estrogen,
TSPs or other glial derived factors on synaptic activity in hESCderived neural networks. Studies summarized in the previous sections strongly suggest a significant contribution of glial derived
factors in developing spontaneous synaptic activity in non-human
primary neural cultures. In future studies, addition of some glial
derived factors such as cholesterol, estrogen, and/or TSPs may be
an alternative to adding xenogenous astrocytes to induce spontaneous events in hESC-derived neurons.
3.4. Role of Glia and Glial-Derived Factors in Formation of
hESC–Derived Neural Networks
Very few studies have demonstrated hESC-derived neural networks with functional synapses and spontaneous activity. The limited success in generating functional hESC-derived neural networks
could be due to the time lag in glial differentiation. In vitro differentiation of hESC to neural lineage first gives rise to neurons and
then glia [13, 16, 43]. Studies have shown that it takes 7- 9 weeks
until astrocytes (GFAP+ cells) appear in neural cultures in vitro [13,
16]. The localization of synaptic proteins and spontaneous synaptic
transmission in neurons develop after astrocytic differentiation.
Until now only a few studies have demonstrated the generation
of spontaneously active neural networks through directed differentiation of hESCs without addition of xenogenous cell types for enhancement [13, 15]. Addition of xenogenous mouse cortical astrocytes to hESC-derived neural cultures resulted in increased synapsin-1 staining (more than 93% of MAP2+ neurons) by 6 weeks in
culture. Moreover, spontaneous synaptic activity was observed as
early as 1 week after plating. Surprisingly, addition of astrocyticconditioned media (ACM) from mouse astrocytic culture did not
enhance synaptic transmission as compared to untreated cultures,
although it increased synapsin-1 puncta and dendritic protrusions.
Failure of ACM to enhance synaptic could be due to species differ-
4. NEUROTROPHINS AND SYNAPSE FORMATION IN
PRIMARY AND ESC-DERIVED NEURONAL CULTURES
4.1. Introduction
Neurotrophins (NTs) are a family of neuronal growth factors
secreted by glia, neurons and/or their target cells. NTs have diverse
biological functions and control neuronal survival, proliferation,
migration, axonal and dendritic outgrowth, differentiation, and
synaptogenesis in neurons. NTs also play important roles in activity-dependent forms of synaptic plasticity in the CNS [44]. Several
NTs have been identified, including nerve growth factor (NGF),
brain-derived growth factor (BDNF), neurotrophin-3 (NT3), and
neurotrophin-4/5 (NT4/5). NTs exert their influence through a variety of signaling cascades by binding to two distinct classes of
transmembrane cell surface receptors, the tropomyosin-related
kinases (Trk)-receptor family and the structurally unrelated p75
neurotrophin receptor (p75NTR, a member of the tumor necrosis
factor receptor superfamily) [45]. Different Trk receptors have different neurotrophin binding affinities. TrkA preferentially binds to
NGF, TrkB binds to BDNF and NT4/5, and TrkC binds to NT3
(TrkA and TrkB bind NT3 to a lesser extent) [46].
The NTs are synthesized and packaged into vesicles in neuronal
cell bodies and transported to pre-synaptic axon terminals and postsynaptic dendrites for local secretion [46]. Neural activity plays an
256 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3
important role in synthesis, transport, and release of NTs. In neuronal cultures, application of glutamate, elevated extracellular K+,
and high frequency external stimulation lead to neuronal depolarization and increased transcription of BDNF mRNA and subsequent
BDNF secretion [47, 48]. In contrast, GABA-mediated inhibition of
neuronal activity decreases BDNF mRNA in hippocampal cultures
[49]. NT binding to Trk receptors results in receptor dimerization
and autophosphorylation, which activates tyrosine-kinase and initiation of one or more signal transduction cascades, including mitogen-activated protein kinase (MAPK), phosphoinositol 3 (PI3)kinase and phospholipase C- signaling pathways. These signals are
either propagated to the nucleus where they influence transcription
factors and alter gene expression or induce changes in the structure
and function of local proteins at synaptic level [46].
4.2. Role of Neurotrophins in Pre-Synaptic Terminal Formation
and Stabilization in Primary Cultures
Neurotrophins (NTs) have strong modulatory effects on the
growth and stability of dendrites and axons in rat hippocampal,
cortical, and cerebellar neurons [50-53]. NTs increase the branching
and complexity of axonal and dendritic arbors, and hence increase
the number of potential contact sites between pre- and post-synaptic
neurons. Following the establishment of nascent connections, NTs
and Trk receptor signaling actively promote the maturation and
stabilization of CNS synapses via pre- and post-synaptic mechanisms.
During synaptogenesis, retrograde Trk signaling modulates the
development of pre-synaptic terminals, in part by regulating the
expression and distribution of synaptic vesicle (SV) proteins (reviewed in [54]). BDNF and NT3 treatment in dissociated and sliced
rat hippocampal cultures result in an increase in the number of SVs
docked at the active zones [55, 56]. Consistent with these results,
BDNF treatment increases the expression of synaptobrevin, a SV
protein, in neuronal cultures [57-59]. Mice deficient in either BDNF
or TrkB receptor exhibit a significant reduction in the total number
of docked vesicles at the hippocampal synapses and a redistribution
of vesicles to areas far from the active zone [60-62]. Moreover,
hippocampal tissue sections from TrkB-deficient mice exhibit an
overall reduction in syntaxin1 and SNAP25 immunoreactivity, and
decreased density of SVs compared to wild-type littermates [60].
As mentioned earlier, syntaxin and SNAP25 are SNARE complex
proteins that are essential for SV fusion and neurotransmitter release. Hence, BDNF plays an important role in regulating synaptic
transmission by regulating expression of SNARE proteins.
The mechanisms by which neurotrophins enhance the formation
and function of pre-synaptic terminals are still unclear. NTs might
modulate either the assembly or the stabilization of synaptic vesicles though post-translation modifications of SNARE complex
proteins [58, 61, 63]. This is supported by the evidence that BDNFdeficient mice exhibit decreased synaptosomal levels of the vesicular proteins synaptobrevin and synaptophysin, although levels from
whole hippocampal extracts appear to be similar to controls [61].
Thus, NTs might regulate vesicle localization and properties of
vesicular release by altering the interactions between SNARE proteins and other vesicular proteins.
4.3. Role of Neurotrophins in Post-Synaptic Modulation in
Primary Cultures
Neurotrophins are emerging as key regulators of post-synaptic
specializations, in addition to their roles in establishing pre-synaptic
terminals and regulating neurotransmitter release. Prolonged exposure to NTs increases the excitatory and inhibitory synapses in
postnatal neural networks by modulating post-synaptic receptors
[64, 65]. In rat neocortical neurons, BDNF promotes the maturation
of silent glutamatergic synapses by increasing the translocation of
the GluR2 AMPA-receptor subunit to the neuronal surfaces [66,
67]. In rat hippocampal neurons, BDNF rapidly increases the phos-
Dodla et al.
phorylation and potentiates the conductance of NMDA receptors
[68, 69]. In inhibitory synapses of rat hippocampal neurons, BDNF
treatment increases the expression of GABAA-receptor subunits,
regulates GABAA-receptor recruitment of the neuronal surface, and
modulates GABAA-receptor channel conductance. Manipulation of
TrkB-mediated signaling results in dramatic changes in the number
and localization of the post-synaptic NMDA-receptor (NMDAR)
and GABAA-R clusters. BDNF treatment increases the number of
NMDAR and GABAAR clusters and increases the proportion of
clusters that are opposed to pre-synaptic terminals, which is decreased by down-regulation of TrkB signaling [46]. These studies
demonstrate that NTs might modulate the synaptic integrity and
strength by modulating the expression, distribution and kinetics of
post-synaptic neurotransmitter receptors at excitatory and inhibitory
synapses.
4.4. Role of Neurotrophins in Developing Spontaneously Active
Networks from Neural Stem Cells
Studies in primary mammalian neuronal cultures have clearly
shown that NTs are essential for neuronal survival, proliferation,
migration, axonal and dendritic outgrowth, differentiation as well as
synaptogenesis. Hence it is not surprising that most of the protocols
for deriving neurons from ESCs use NTs (for example [13, 15, 70]).
However, there is lack of studies looking exclusively at effects of
NTs on synapse formation on ESC-derived neurons. Most of our
knowledge comes from studies in primary mammalian neuronal
cultures and neural stem cell cultures.
Studies which have demonstrated functional neural network
formation in mESC- and hESC-derived neurons have used NTs in
the culture media [13, 15, 16, 70]. However, it is not clear from
these studies if NTs are essential for this derivation, or, how would
removal of NTs effect functional synapse formation. Studies comparing the effect of NTs on functionally active neural networks
from hESC-derived neurons have not been published yet. However,
as mentioned in previous sections, studies in neural stem cells
(NSC) and primary neurons clearly suggest that NTs alone or in
synergism with CAMs are essential for functionally active synapse
formation. In future studies with hESC-derived neurons, role of
NTs in the localization of SV and SNARE proteins and formation
of functional synapses holds great promise.
5. CELL ADHESION MOLECULES AND DIFFUSIBLE FACTORS IN PRIMARY AND HESC-DERIVED NEURONS
SYNAPTOGENESIS
Synaptogenesis is a multistep process involving several signaling molecules. The initial contact between a neuronal axon and its
target cell is followed by establishment of stable sites of cell-tocell-contact, followed by pre- and post-synaptic differentiation.
Both processes seem to be influenced to some degree by various
classes of cell adhesion molecules (CAMs) and diffusible factors.
5.1. Priming Molecules in Synapse Formation in Primary
Cultures
Several diffusible factors and CAMs have been implicated in
target recognition and initial processes of synaptogenesis. Some of
the diffusible factors and CAMs have been termed as “priming
molecules” because they seem to prime neurons and make them
competent to form synapses but cannot promote formation of mature synapses by themselves. The priming molecules promote axonal and dendritic maturation, and/or accumulation of recycling
synaptic vesicles in innervating axons, and facilitate the ability to
initiate synaptogenesis [71]. Priming molecules include members of
the cadherin family of calcium-dependent cell adhesion molecules,
including cadherins and protocadherins, and diffusible factors secreted by neurons and glia [71].
The classical cadherins, which are homotypic CAMs, are individually localized to pre- and post-synaptic membranes in a variety
Role of Astrocytes, Soluble Factors, Cells Adhesion Molecules
Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3
257
of synapses at early stages of synaptogenesis [72, 73]. However,
studies indicate that classical cadherins are not directly involved in
triggering synapse formation. For example, blocking N-cadherin
mediated cell-cell contact using antibodies in the developing chick
optic tectum causes retinal ganglion cell axons to overshoot their
targets and form ectopic synapses but does not inhibit synapse formation per se [74]. Results from similar studies suggest that cadherins play a role in target specification and perhaps stabilization of
early synaptic contact sites but not in the induction of synapse formation [75]. Protocadherin, a second class of CAMs, have also
been shown to be involved in target recognition and synapse specification. Similar to classical cadherins, protcadherin-gammas partially localize to synaptic sites [76], and genetic studies in Drosophila indicate that they are involved in target recognition rather than
synapse formation [77]. Studies of protocadherin knockout mice
support this conclusion and indicate that these CAMs are not essential for neuronal differentiation or synapse formation but rather for
stabilization of synapses and neuronal survival [78].
Diffusible factors including members of the Wnt and fibroblast
growth factor (FGF) families have also demonstrated roles in synaptogenesis in primary neural cultures. These factors act by inducing arborization of axons, clustering of SV associated proteins, and
formation of pre-synaptic active zones. Some of these factors with
known functions are: Wnt-3 [79], Wnt-7a [80], FGF22 with FGF2
[81], FGF7 and FGF10 [81]. However, none of these factors are
sufficient to form mature synapses by themselves.
domains of pre-synaptic scaffolding proteins such as CASK (calcium/calmodulin dependent serine protein kinase). In turn CASK
helps create a protein complex that includes N-type voltage gated
Ca2+ channels. Similarly, in the post-synaptic cell, cytoplasmic
domain of neuroligin contains PDZ-binding motifs that bind to PDZ
domains of the PSD protein SAP90/PSD95, which in turn can bind
to subunits of NMDA receptors and other molecules. These findings have led to the hypothesis that neurexin-neuroligin complexes
play an important role in maintaining the pre-synaptic SV release
machinery and post-synaptic reception machinery. Moreover, neuroligin expressed by non-neuronal cells, HEK293, led to induction
of functional pre-synaptic structures along axons of pontine neurons
growing over the HEK293 cells, suggesting that neuroliginneurexin interactions might be important during synaptogenesis
[89].
SynCAM (synaptic cell adhesion molecule) is a cell-surface adhesion molecule capable of inducing pre-synaptic differentiation.
SynCAM is a member of the Ig superfamily of adhesion molecules
and a homophilic CAM expressed on both sides of the synapse [90,
91]. SynCAM1 over expression in cultured neurons results in enhanced synapse formation; whereas, over expression in the nonneuronal cells results in formation of functional pre-synaptic active
zones in axons contacting these cells [90]. Moreover, expression of
dominant-interfering construct against SynCAMs compromised
pre-synaptic differentiation [90]. These studies indicate that SynCAM is a potent inducer of pre-synaptic differentiation in neurons.
5.2. Inducing Molecules in Synapse Formation in Primary
Cultures
Several classes of molecules, termed “inducing molecules”, are
capable of directly inducing various aspects of synapse formation.
These include Neuronal activity-regulated pentraxin (Narp) and
EphrinB1, two secreted proteins capable of clustering subsets of
post-synaptic proteins, and SynCAM and Neuroligin, two CAMs
that can trigger the formation of functional pre-synaptic terminals.
Narp is a member of the pentraxin family of secreted proteins
that localizes to synapses and also binds to the extracellular domains of subunits of the AMPA-type glutamate receptor. When
over expressed in rat spinal cord neurons, Narp increases the synaptic clustering of AMPA receptors [82]. Further, when rat spinal
cord neurons were co-cultured with Narp-expressing HEK293 cells,
it lead to clustering of surface AMPA receptors co-expressed with
Narp on HEK293 cells, as well as ectopic clustering of neuronal
AMPA receptors on rat spinal cord neurons [83]. Furthermore,
Narp promotes clustering of NMDA as well as AMPA receptors in
certain classes of interneurons [84]. These data suggest that Narp
has potent ability to induce AMPA and NMDA receptor clustering
at glutamatergic synapses.
EpherinB, a member of the Epherin family of axonal growth
cone guidance molecules, also has been shown to have synaptogenic activity. EpherinB family members interact with their receptor, EphB, which in turn interacts with and promotes clustering of
NMDA-type of glutamate receptors in rats [85]. Epherins and Eph
receptors have also been implicated in dendritic spine development
[86] and maturation [87]. Interestingly, EpherinB-mediated aggregation of EphB receptors does not induce co-aggregation of other
post-synaptic components such as PSD-95 family proteins, scaffolding proteins that normally aggregate along with NMDA receptors. This suggests that the EpherinB-EphB interactions regulate
some but not all aspects of post-synaptic differentiation.
Post-synaptic neuroligins and its pre-synaptic receptor neurexins and are CAMs with potent ability to induce synapse specification, adhesion and signaling during synaptogenesis [71]. Neurexins,
expressed in two principal forms, - and ß-neurexins, are highly
polymorphic due to extensive alternative splicing. Neuroligins bind
to and induce clustering of -neurexin [88]. The cytoplasmic domain of neurexins has PDZ-binding motifs that can bind to PDZ-
5.3. Cell Adhesion Molecules and Diffusible Factors in ESCDerived Neuronal Synaptogenesis
In this section we summarize the studies that have identified
roles of CAMs and diffusible factors in synapse formation in ESCderived and progenitor cell-derived neuronal cultures. N-cadherin is
a synaptic cell adhesion molecule localized perisynaptically in the
vicinity of the active zone and the PSD of glutamatergic synapses.
Jüngling et al. studied the functional role of N-cadherin in glutamatergic synapses by studying the synapse formation in cultured Ncadherin knock-out, ESC-derived neurons [92]. Interestingly, initial
synapse formation was not affected in N-cadherin knock-out neurons, as indicated by the density of Synapsin I puncta, the density of
FM4-64 puncta, the basic ultrastructure of asymmetric synapses,
and the presence of post-synaptic AMPA and NMDA receptormediated currents. However, drastic defects in synaptic function
were observed under conditions of high synaptic activity in Ncadherin knockout neurons. There was reduction in
K+ depolarization-stimulated AMPA receptor mediated miniature
EPSCs and reduced efficiency of vesicle exocytosis during high
synaptic activity, as compared to wild-type neurons. Together, these
results indicate a reduced availability of vesicles during high synaptic activity, and that N-cadherin plays an important role in regulating synaptic function during high synaptic activity. In rat hippocampal progenitor cells, addition of neural cell adhesion molecule
(NCAM) inhibited neuronal progenitor cell proliferation and increased the number of neurons generated [93]. Addition of FGF2
and either N-CAM or BDNF differentiated the cells into spontaneously active neural networks which displayed characteristic action
potentials when cultured on multi-electrode arrays (MEAs). Individually, N-CAM, BDNF or FGF2 did not result in spontaneous
activity, indicating that synergism between N-CAM and FGF2, or
between BDNF and FGF2 is essential for spontaneous activity. This
study indicates that growth factors can synergize with neurotrophins or N-CAM to generate spontaneously active neural activity
from rat hippocampal progenitor cells.
Apart from the two studies mentioned above, there is a dearth
of studies on the effects of diffusible factors and CAMs on synaptogenesis in progenitor cell-derived and ESC-derived neurons. As
summarized in the previous sections, there is wealth of information
on the roles of various CAMs and diffusible factors in synapse
formation in primary cultures; however, very few studies have
258 Current Stem Cell Research & Therapy, 2010, Vol. 5, No. 3
mation in primary cultures; however, very few studies have looked
at their roles in ESC-derived neurons, in particular hESC-derived
neurons. In primary neuronal cultures, addition of diffusible factors
such as FGF22, FGF2, Wnt3, Wnt7a, and EphrinB promote some
aspects of synaptogenesis. Similarly overexpression of CAMs such
as cadherins, protocadherins, Neuroligins-neurexins, and SynCAM,
promotes synaptogenesis in primary cultures. In future studies with
ESC-derived neurons, similar strategies using a combination of two
or more of these factors might result in enhanced synaptogenesis
and formation of spontaneous neuronal networks.
CONCLUDING REMARKS AND FUTURE DIRECTIONS
FOR ESC-DERIVED NEURONS
Until recently, there was lack of studies demonstrating hESCderived neurons able to form spontaneously active neural networks
and functional synapses. Only recently two publications have demonstrated such capabilities. In both of these studies it took up to 7- 9
weeks for the networks to demonstrate mature action potentials and
burst activity. The long time it takes for the neural network to exhibit spontaneous network activity and lack of enough published
studies clearly highlight the difficulties in achieving this goal.
In this review article we discuss several factors that promote
synapse formation in primary neuronal cultures and neural stem
cells. In future studies addition of these factors to hESC-derived
neuronal cultures might enhance functional synapse formation.
Astrocytes and astrocyte-derived factors such as, cholesterol, estradiol, thrombospondins, and glutamate significantly modulate excitatory synapse formation between primary neurons. Although addition of rat primary astrocytes significantly enhances synapse formation in hESC-derived neuronal networks, it introduces a xenogenous cell which is not desirable. Hence addition of factors such as
cholesterol, estradiol and thrombospondins to hESC-derved neuronal cultures is more desirable. Similarly addition of neurotrophins
and over expression of CAMs in neurons might lead to faster maturation of neuronal synapses. All of the aforementioned factors are
presented in a strict spatial and temporal sequence in vivo. Hence it
might be necessary to present some of these factors in a temporal
sequence. For example, certain CAMs might be first needed for
synapse specification, followed by other CAMs, neurotrophins and
astrocytic-factors for synapse maturation. Addition of some of these
factors might lead to formation of synaptically active neural networks. hESC-derived neurons hold great promise for drug screening studies, cell replacement therapy and developmental studies,
which might remain unfulfilled if the neurons fail to establish mature functional synapses. The factors we mention in this review
offer new approaches to enhance functional synapse formation and
help hESC-derived neurons realize their full potential.
ACKNOWLEDGEMENTS
We thank Ms. Amber Young for critical reading of the manuscript. We also acknowledge the funding support of Department of
Defense grant N000140810989.
ABBREVIATIONS
ABP
= AMPA receptor binding protein
AMPA = -amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid
AZ
= Active zone
BDNF = Brain derived neurotrophic factor
CAM
= Cell adhesion molecule
CASK = Calcium/calmodulin dependent serine protein kinase
ECM
= Extracellular matrix
FGF
= Fibroblast growth factor
GABA = Gamma aminobutyric acid
GCM
= Glial conditioned media
Dodla et al.
GFAP
=
GK
=
GKAP =
GRIP
=
hESC
=
LDL
=
MEA
=
mESC
=
MAGUK =
MAPK =
Narp
=
NCAM =
NMDA =
NGF
=
NSF
=
NT
=
PI3
=
PICK 1 =
PSD
=
RGC
=
RIM
=
SAP90 =
SNAP-25 =
SNARE =
SV
=
SynCAM =
TNF-a
=
Trk
=
TSP
=
TTX
=
VAMP2 =
Glial fibrillary acidic protein
Guanylate kinase
Guanylate kinase associated protein
Glutamate receptor interacting protein
Human embryonic stem cells
Low density lipoprotein
Multi-electrode array
Mouse embryonic stem cell
Membrane associated guanylate kinase
Mitogen activated protein kinase
Neuronal activity-regulated pentraxin
Neural cell adhesion molecule
N-methyl-D-aspartate
Nerve growth factor
N-ethylmaleimide-sensitive fusion protein
Neurotrophin
Phosphoinositol 3
Protein interacting with C kinase 1
Post-synaptic Density
Retinal ganglion cell
Rab3 interacting molecule
Synapse associated protein-90
Synaptosomal associated protein
Souble N-ethylmaleimaide sensitive factor attachment protein receptors
Synaptic vesicle
Synaptic cell adhesion molecule
Tumor necrosis factor-alpha
Tropomyosin-related kinases
Thrombospondin
Tetrodotoxin
Vesicle associated membrane protein 2
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Revised: December 22, 2009
Accepted: March 20, 2010