Glutamate Induced Calcium Signals in
Proliferating Cells of the
Central Nervous System in Adult Zebrafish
Philip Merksamer
Advisor: Stuart Thompson
June 2003
Permission is granted to Stanford University to use the citation and abstract of this paper.
Abstract
The periventricular zone (PVZ) of the adult zebrafish central nervous system contains
undifferentiated, proliferating cells consistent with the properties of neural progenitors. Past
studies suggest that axonal projections may extend from the optic tectal neurons into the PVZ
allowing for the possibility that the optic tectal neurons signal the PVZ with neurotransmitters,
In this study, time-lapse confocal imaging of PVZ cells labeled with fluo-4 AM calcium
indicator is employed to demonstrate that PVZ cells respond to glutamate through elevated
intracellular calcium levels. The NMDA receptor antagonist, APV, and the AMPA receptor
antagonist, CNQX, block this response. PVZ cells appear to express functional AMPA and
NMDA receptors, and these receptors may play an important role in proliferation, migration, or
differentiation of the PVZ cells.
Introduction
Neural progenitors are immortal cell lines capable of differentiating into mature neural
tissue. Considerable amounts of research are devoted to the study of neural progenitor cells in
order to elucidate the mechanism of neural regeneration as well as to develop treatments for
neural degenerative disorders. Zebrafish (Danio Rario) are advantageous model organisms for
such research because they generate new neurons throughout their adult lives and thus are
presumed to posses neural progenitors (Thompson, unpublished data).
Past studies indicate that the round cells of the periventricular zone (PVZ) in the optic
tectum of adult zebrafish may be such progenitor cells. These cells have been shown to
proliferate both in-vivo and in culture (Tulloch, 2002; Webster, 2002).
While the morphology of the PVZ cells has been well characterized, little is known about
signaling and activation of these cells. There are indications that axonal projections extend from
the optic tectum into the PVZ permitting the possibility that optic tectum neurons interact
synaptically with the PVZ cells (Arakaki, unpublished data).
Synaptic interaction between neurons is crucial for proper CNS function. It is unknown,
however, whether synaptic interaction between neurons and neural progenitors exists, and
whether such interaction is necessary for neural progenitor activation and differentiation.
This study addresses the question; do PVZ cells respond to the neurotransmitter,
glutamate, through NMDA- and AMPA-type glutamate receptors? Glutamate signaling acting
on both NMDA receptors (NMDA-R) and AMPA receptors (AMPA-R) has been shown
necessary for proper development of dendritic arbors in Xenopus optic tectal neurons (Rajan &
Cline, 1998). A similar mechanism may operate in the signaling of PVZ cells in zebrafish.
In this study, time-lapse confocal images were taken of living optic tectum slices during
the application of glutamate and glutamate receptor antagonists. These images were analyzed for
increased intracellular calcium concentration using the fluorescent indicator, fluo-4 AM. An
elevated intracellular calcium concentration is indicative of a response to glutamate because both
AMPA-R and NMDA-R generate a rise in intracellular calcium.
I find that the PVZ cells respond to glutamate through elevated intracellular calcium
levels. This response is blocked by the selective NMDA-R antagonist, APV, and by the selective
AMPA-R antagonist, CNOX.
Methods
Brain Slice Preparation. Adult zebrafish brains were dissected in Hanks media containing
(mM): Nacl 96, KCI 3.78, NazHPO4 0.175, KH2PO4 0.308, CaClz 0.91, MgSO4 0.7, NaHCO;
4.2, CeHj2O6 25. The optic tectum lobes were cut from the brain and incubated with SuM of the
calcium sensitive indicator, fluo-4 AM (Molecular Probes) for forty minutes, followed by a ten
minute wash with either Hanks or Hanks plus the glutamate receptor antagonists APV (100 uM)
or CNQX (100 uM).
Confocal Microscopy. Time-lapse confocal images were taken of optic tectum slices using an
Olympus microscope with a 40x water-immersion objective. Äfter the first ten frames, 1 ml of
Hanks, followed by 1 ml of 10 uM glutamate, or 1 ml of Hanks, followed by 1 ml of 10 uM
ionomcyin was added to the profusion dish containing the optic tectum slice. lonomycin is a
calcium sensitive ionophore and was used to determine the saturation point for the fluo-4 AM
indicator. Images were taken for a total of 120 frames, with approximately two seconds between
each frame.
Fluorescence analysis. Äfter imaging, six to ten regions of interest were sampled for
fluorescence intensity vs. time. Baseline fluorescence, Fo, was defined as the average
fluorescence over the first 10 frames. Fluorescence was averaged over 5 pixels and converted to
units of AF/Fo. Fluorescence intensities were converted to calcium concentration (nM) from the
following equation:
Ca2 - Ka' (F - Fmin)/(Fmax - F)
Kq, the dissociation constant for fluo-4 AM, was 345 nM. Fmax was given by the response to
ionomcyin and was set to 1.6. Fmin was set to 0.826 so that an F value of 1 gives a calcium
concentration of 100 nM.
Results
The optic tectum slices were fully saturated with fluo-4 AM indicator after incubation for
one hour (Fig. 1), however, a forty minute incubation was sufficient for confocal imaging. The
indicator stained only the edge of the tectum and the periventricular zone closet to the edge.
The addition of the calcium specific ionophore, ionomycin, resulted in an elevated
fluorescence intensity from 1 to 1.4 AF/F (Fig 2). The intracellular calcium concentration
increased from a resting concentration of 104 nM to 1058 nM.
The addition of glutamate resulted in elevated fluorescence intensity compared to a saline
control reaching a maximum intensity of approximately 1.27 AF/F (Fig 3). The intracellular
calcium concentration increased from a resting concentration of 102 nM +- 38 (95% confidence
interval) to 366 +- 149 nM averaged over 16 individual cells from two independent experiments.
When the optic tectum slice was incubated with the NMDA-R antagonist, APV, the
addition of glutamate resulted in no elevated fluorescence intensity compared to a saline control
(Fig 4). The calcium concentration increased from 100 +- 37 nM to 112 +- 38 nM averaged
over eight cells.
When the optic tectum slice was incubated with the AMPA-R antagonist, CNQX, the
addition of glutamate resulted in a slight elevated fluorescence intensity compared to a saline
control reaching a maximum intensity of 1.12 AF/F (Fig 5). Calcium concentration increased
from 100 +/- 44 nM to 160 +- 76 nM averaged over 18 cells from two independent experiments.
Discussion
In this study, I have provided evidence that the neural progenitors of the PVZ express
functional ionotropic glutamate receptors. First, I demonstrated that the PVZ cells respond to
glutamate through a 3-fold increase in intracellular calcium. Second, I found that the selective
antagonists, APV and CNQX blocked this elevated calcium response. Because APV and CNOX
specifically block NMDA-R and AMPA-R respectively, the cells in the PVZ contain both types
of receptors.
There is considerable evidence that embryonic mammalian cells within ventricular zones
(VZ) express many amino acid neurotransmitters and their respective receptors (Lo Turco et al.,
1991 & 1995). However, not all mammalian VZ cells proliferate, and some precursor cells begin
differentiation in the ventricular zone (Li et. al., 1998). Furthermore, using a calcium imaging
technique similar to this study, Maric et. al. found evidence that AMPA/kainate receptors first
appear in the terminal cell division stage of embryonic neural precursor cells and may be
involved in differentiation and commitment to a neuronal cell lineage (2000).
In this study, there was considerable variability in the glutamate response of PVZ cells
(366 + 149). This variability could have a physiological basis in that not all the cells of the
PVZ may express ionotropic glutamate receptors. It is possible that only a subset of PVZ cells
that have begun to differentiate are expressing such receptors which would be consistent with
previous findings in mammalian systems. It is also possible, however, that the variability was
due to the experimental design. The quality of staining with fluo-4 AM was not consistent, and
sometimes the PVZ cells would not take up the indicator.
This study could be bolstered with more experiments, particularly one using the agonists,
AMPA and NMDA. An elevated calcium response to AMPA and NMDA would provide strong
supporting evidence that the PVZ cells express functional AMPA and NMDA receptors. Also,
an in-situ hybridization analysis for mRNA transcripts encoding AMPA and NMDA submit
genes would furnish further evidence that these receptors are actively expressed.
Finally, the extent to which the ionotropic glutamate receptors are involved in
proliferation, migration, and differentiation in the PVZ cells remain unknown. However, the
existence of such receptors and the fact that they cause elevated cytosolic calcium levels implies
that glutamate acting on ionotropic receptors has a considerable role in PVZ cell signaling.
Conclusion
Using time-lapse confocal imaging, this study demonstrated that the PVZ cells considered to be
neural progenitors respond to glutamate through elevated calcium levels. The antagonists, APV
and CNQX, blocked this response, indicating that the PVZ cells express functional NMDA- and
AMPA-receptors. The ionotropic glutamate receptors thus appear to be involved in cell
signaling in the PVZ, however it is still unknown whether these receptors are directly involved in
proliferation, migration, or differentiation, three critical features of progenitor cells.
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Appendix 2: Figures
Normalized Average Intensity vs. Time After Dye Loading
10
Time (10 Minute Intervals)
Figure 1: Average intensity vs. Time after dye loading.
AF/Fvs. Time
1.6
1.4
— Control
1
lonomycin 1OuM

0.9
0.8
150
200
100
50
time (sec)
Figure 2: AF/F vs. Time for the Negative Control (saline) and the positive control (ionomycin).
Calcium Response to Glutamate
— Contro
Glutamate10UM

0.9
0.8 -
50 100 150 200 250 300
time (sec)
Figure 3: AF/F vs. Time for the negative control and glutamate.
Calcium Response to Glutamate in the
presence of APV (T00uM)
1.6
1.4
+ 1.3
— Contro
Glutamate 10UM

0.9
0.8 -
50 100 150 200 250 300
time (sec.)
Figure 4: AF/F vs. Time for control and glutamate in the presence of APV.
Calcium Response to Glutamate in the
Presence of CNOX (100uM)
— Control
Glutamate 10uM
lonomycin 10uM


0.9
0.8
0
50 100 150 200 250 300
time (seo
Figure 5: AF/F vs. Time for control, glutamate, and ionomycin in the presence of CNOX.
600
500
400
Conto
Glutamate
300
□ Glutamate with APV
EGlutamate with CNOX
100
Figure 6: Bar Graph comparing calcium concentration for each treatment.