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Biotempo 2014, Volumen 13, 39-45
ChARACTERIZATION OF GLUTAMATERGIC PhENOTYPES IN
hYBRID SEPTAL NEUROBLASTOMA (SN56) CELLS
Luis F. Pacheco
1
M. Velazquez
1
M.Villarreal
1
J. Rodriguez
1
Boris Ermolisnky
1.2
Emilio R. Garrido-Sanabria
1,
ABSTRACT
septum neurons (cholinergic) and tumoral neuroblastoma cells. Cholinergic cells synthesize and release the
the vesicular glutamate transporter type 1 (VGluT1), a protein that is normally produced by glutamatergic
phenotype which is important because glutamatergic neurons have been associated to the pathogenesis of
expression of glutamatergic phenotype, by qPCR, western blotting and Immunocytochemistry assay. The
were separated by each experiment, primary antibodies or primers against NMDA glutamate receptor subunit
hypothesis,. Expression of these markers will indicate a glutamatergic phenotype. After secondary detection
glutamate NR2B receptor subtype and the VGluT1 transporter in both post-synaptic and presynaptic structures
and glutamatergic phenotype.
1
Department of Biological Sciences at the University of Texas at Brownsville/Texas Southmost College, Brownsville, Texas 78520 USA.
INTRODUCTION
The murine basal forebrain cholinergic cell line
neurons of the mouse postnatal day 21 septum
and the murine neuroblastoma cell line N18TG2
cholinergic neuronal phenotype upon stimulation
with membrane permeable dibutyryl cAMP
(dbcAMP) and all-trans-retinoic acid (Blusztajn et
1995) and nerve growth factor (Nilbratt et al., 2007).
Cholinergic properties include: increased synthesis
of acethylcholine (Ach), enhanced activity of
choline acetyltransferase (ChAT), spontaneous and
depolarization-evoked ACh release (Blusztajn et al.,
upregulation of vesicular acetylcholine transporter
(VAChT) gene expression (Berse and Blusztajn,
1995), increased expression of different nicotinic
acetylcholine receptor (nAChR) subtypes (Nilbratt
et al., 2007) and several morphological features
protein immunoreactivity, puncta adherens,
neuritic varicosities, vesicles) (Hammond et al.,
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2
The Center for Biomedical Studies
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Biotempo 2014, Volumen 13, 39-45
to investigate the pathogenesis of neurological
characterized by cholinergic dysfunction and
progressive basal forebrain cell loss (Mufson et
characteristics that make it a useful model for
studying neuronal differentiation. This cell line,
when treated with Retinoic Acid and cAMP, stops
therefore, it makes it into an ideal cell line to study
neuronal function. It is not clear whether these cells
form excitatory glutamatergic synapses. In this
study, we investigate this problem by performing
electrophysiology, calcium imaging, qPCR and
immunocytochemistry and co-localization analysis
marker of glutamatergic synapses. The antibodies
work while investigating the excitatory synapses
expressing glutamatergic markers.
receptive to glutamatergic synapses, we will be
the expression of glutamatergic markers including
vesicular glutamate transporter1 (VGluT1) and
glutamate receptor subunit epsilon-2 (NR2B) in
MATERIALS AND METhODS
1. Cell Culture
Hammond et al., 1990) previously shown to express
that was generously provided
(Department of Pathology, Emory University
School
of Medicine, Atlanta, GA). The cells were
atmosphere of 5%
2
Sigma-Aldrich, Saint Louis, USA) supplemented
with
10% fetal bovine serum and penicillin-
cells were passed at 1:10
were cultured in serum-free DMEM supplemented
density of ~1×10
cells on coverslip-lysine of 12 round diameter were
10% fetal bovine serum with 1 mM glutamine and
100ug/ml penicillin/streptomycin per 1 ml at 37 °C
2
. The cells
were differentiated by 1, 2, 3 and 4 days with 1mM
ibutyryl-cAMP (cAMP)/1uM
all
trans-Retinoic
acid (RA), or no-differentiated (control)
2. Immunocytochemistry
Following the plating of both differentiated (using
15 min with 4% paraformaldehyde/PBS followed
by 3 PBS washes. Antibodies(Ab) were added:
mouse monoclonal Ab 75-097, (NR2B), rabbit
polyclonal Ab 32942 (NMDA)and guinea pig
,
polyclonal Ab 5905 (vGlut1). Then, they were left
to incubate overnight. After washing the cells three
times with PBS they are exposed to corresponding
secondary antibodies. Finally, the cells are washed
three times with PBS and mounted to a slide prior
to viewing. Imaging was performed using laser
inverted microscope.
3. qPCR.
Real-time PCR reactions were carried out
mGlu1/5, NR2B and nAChR previously validated
in end-point PCR assays. The data was collected
was performed using the comparative threshold
(CT) method after determining the CT values for
reference () and target genes.
GAPDH
4. Calcium Imaging
Intracellular Ca
2+
measure-
ments using Fluo-4 Direct Calcium Assay Kits (in-
were treated with 1 µM probenesic at time zero and
-
lation. Positive control was used ACSF
RESULTS AND CONCLUSION
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with 1 mM dibutyrylcyclic AMP (dbcAMP) for 24,
demonstrate the co-existence of the pre-synaptic
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Biotempo 2014, Volumen 13, 39-45
Vesicular Glutamate Transporter 1 (vGlut1) with
the post-synaptic Glutamate Receptor Subunit
Epsilon-2 (NR2B) and the metabotropic glutamate
results electrophysiological, faster action potential
addition the ratio the expression in differentiated
mGlu1/5, NR2B and nAChR in no differentiated
cells.
This cell line model will now be used to further
investigate drugs that modulate excitatory glutamate
transmission along with cholinergic transmission
with potential implications for understanding the
basic mechanisms of epilepsy and other neurological
disorders.
Acknowledgments
This work was supported by grants from LSAMP and
National Institutes of Health as follows: NS063950-
03, 3SC1NS063950-03S1 (ARRA), P20MD001091,
P20MD000161 to Dr. Garrido-Sanabria by the
National Institute of Neurological Disorders., ARRA
3R25GM065925-06S1
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A
. Current
clamp recordings of undifferentiated cells shows wide action potentials in response to electrical stimulation
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Biotempo 2014, Volumen 13, 39-45
(square current steps, below). . Differentiated neuron exhibited faster action potential in response to similar
B
stimulation protocol. and . Voltage-clamp recordings revealed small sodium current in undifferentiated
CD
cells ( and while differentiated neurons exhibited large sodium currents. . Analysis of the current
C1C2)E
activation kinetics shows that sodium currents in differentiated cells have faster rising and decay phases (
E
)
and are activated at lower membrane potential ().
FG
* Student t-test, <0.05) and current density (normalized to the cell capacitance) (undiff: -25.0 ± 5.5 pA/pF
P
P
<0.05). Time constant of activation was also faster in
differentiated cells (Student t-test, p<0.05).
Immunocytochemistry for Glutamate Vesicle Transporter (vGlut1) and Glutamate Receptor
Figure 2: (A) Represents the validation immunocytochemistry of the polyclonal antibody vesicle transporter
cells
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Biotempo 2014, Volumen 13, 39-45
Co-localization of NMDA with vGlut1 and NR2B
Figure 3:
performed. This chart illustrates the presence of vGlut1, NMDA, and NR2B. NMDA, the glutamate receptor
family in which NR2B belongs to (both post synaptic structures), peaks with NR2B. Next, you see the peak of
vGlut1, a pre-synaptic transporter.
Figure 4. Relative gene expression analysis of vGlut1, mGlur1, NR2B and nAChR respectively was performed
by the comparative CT qPCR method using primers (see the legend). Data (fold changes) is represented as
arbitrary units normalized relative to gene expression control group. Analysis was performed in total RNA
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Biotempo 2014, Volumen 13, 39-45
-
neurons exhibit spontaneous activity. Pseudoco-
lored image sequence of changes in intracellular
spontaneous activity , monitored at 5 frame per
microscope. A1. Fluorescence intensity changes
over time in neurons 1, 2 and 3 revealed sponta-
neous activity and synchronization of activity bet-
represent increases in intracellular Ca2+ concentra-
tions (Ca2+ transients or spikes) which are related
to spontaneous action potentials in these neurons.
of NMDA (AP-5, 50 uM) and AMPA receptors
activity. B1. Fluorescence intensity changes over
time in neurons 1 and 2. C. Representative bar gra-
ph depicting pool data on changes in event (Ca2+
transients) frequency befor and after pharmacolo-
gical block of NMDA and AMPA receptors. Paired
Primers:
a
. 5-tgcctcaggcttaagatgca-3 Forward
a
. 5-caagcggagaacgactttca-3 Reverse
b
. 5-ccttgcaccgtctgatttggaggctg-3 Forward
b
. 5-cagcgcgaaccaccctgacg-3 Reverse
c
. 5-cccagaccacaagcgctact-3 Forward
c.
5-gcctccactgaccgaatctc-3 Reverse
d.
5-acggcgctttgccattgtc-3 Forward
d.
5-ccagcttcatgctgcagttc-3 Reverse
Legend:
a. vGlut1, b. mGlur1/5, c. NR2B, d. nAChR
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with red representing high Ca2+ concentrations and
blue representing low Ca2+ concentrations. Scale
bar for A and B = 100 microns.
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