Transcript Poster Example

Glia in Cell Culture Insert Support Neurons in vitro
1
SAADIA HASAN ,
and Kelsey C.
2
3
4
Martin
Interdepartmental Program, 2Department of Biological Chemistry, 3Department of Psychiatry and Biobehavioral Sciences, 4Integrated
Center for Learning and Memory, David Geffen School of Medicine, University of California, Los Angeles.
The study of neuron-specific factors requires pure neuronal cultures. However, the absence of glia can affect the
physiology of neurons negatively. This study compared three different treatments of neuronal cultures in order to find
a treatment that reversed the deleterious effects of removing glia from neuronal cultures. The first culture, the control,
consisted of a mixture of plated neurons and glia. The second culture consisted of plated neurons and glia treated
with an antimetabolic agent, cytosine arabinoside, to kill the glial cells. Lastly, the third culture consisted of plated
neurons with a neuron and glia plated cell culture insert, containing a permeable bottom to allow passage of secreted
factors such as modulatory proteins. To compare the effects of the different conditions on synaptic activity, miniature
excitatory post-synaptic currents (mEPSCs) were recorded using a whole-cell voltage clamp and the number of
synapses was monitored using immunocytochemistry. Voltage-clamp data suggested a significant increase in mEPSCs
frequency in the insert condition compared to control, indicating that the neurons were healthy and had robust
synaptic activity in the presence of the insert. Immunocytochemistry experiments showed restoration of the number
of synapses as well as postsynaptic and presynaptic components in the insert to control levels. This indicated that the
insert could rescue the effects of removing glia, although the insert increased synaptic function as compared to
control levels.
a
Results
Control
AraC
Insert
b
Figure 1: Qualitative analysis shows insert rescues cell density. Differential interference contrast (DIC) images
of the plated hippocampal cells from the three conditions. The scale bars represent 100μm. The cell density was highest in the control group, with the
araC group having the lowest cell density.
Merged
Control
AraC
Insert
Figure 4: Neuron morphology is normal in the presence of
the insert. (a) Dendritic segments labeled with antibodies to GluA1 (AMPAr subunit) and
synapsin (presynaptic marker). Synapses were defined as areas where GluA1 and synapsin colocalized. There were fewer synapses in the araC condition, but the insert restored the number of
synapses to control levels. (b) Neurons labeled against MAP2 stain. The branching pattern was
similar in the control and insert groups.
Significance
• Because glia are important for neuronal health and function, pure neuronal cultures
can behave in non-physiological ways.
• Therefore it is important to find a method to provide glial support to pure neuronal
cultures.
• AIM: to evaluate a method, using glial secretions from a cell culture insert, for
studying glia-free neuron cultures without the detrimental effects of getting rid of glia.
GluA1
Control
AraC
Insert
Background
• Glia support neurons in several ways. A few of their functions involve supplying
nutrients1, regulating synaptic transmission1 3 and long term potentiation (LTP)3,
neurotransmitter reuptake1 3 5 and synapse pruning during development1 4.
• Glial processes are closely intertwined with neuronal processes. Even with
sophisticated imaging and separation techniques, this can make it very difficult to tell
if a certain molecule is present in the glial cell or the neuron.
Synapsin
MAP2
1Neuroscience
Klara Olofsdotter
2
Otis
a
Control
b
AraC
Methods
Dissociated hippocampal cultures and treatments:
• Hippocampal cells from P0 mice were plated on a coverslip at a density of 40,000
and inside a cell culture insert (pore size 3 μm) at a density of 60,000 cells per insert.
• Cytosine Arabinoside (araC; 2μM) was used as the antimetabolic agent to kill glia.
• Coverslips were treated as shown below.
Insert
c
Figure 2: Average inter-event
interval and average peak
amplitude of mEPSCs from the
insert group show increased
synaptic activity. (a) representative
recordings of mini-excitatory postsynaptic currents
(mEPSC) from each experimental group showing
increased synaptic activity in insert group. Overall,
the insert group had the highest frequency of
mEPSCs recorded. (b) shows the average interevent intervals (IEIs) of the mEPSCs from the
three groups. The control group had the largest
IEIs, which corresponds to the lowest frequency of
mEPSCs. The insert group had the shortest IEI
(highest mEPSCs frequency), and it was
significantly smaller than the control group. AraC
data were not included in the statistical analysis due
to a small sample size (n=4), however, the group
was included in the figures for comparison. (c)
represents the average peak amplitude of mEPSCs
from each group and, likewise, there was a
significant increase in peak amplitude between the
control and insert group.
Discussion
• The increase in mEPSC frequency in the insert group might suggest either
► more synapses
► that synaptic events are more easily detected at the soma
• Synaptic events could be easier to detect if more AMPA receptors are inserted so
that mEPSC amplitudes are larger, or if synapses are closer to the soma.
• An increase in mEPSC amplitude is also generally taken to represent insertion of
more AMPA receptors.
• However, while immunocytochemistry experiments suggest that the levels of the
AMPA receptor subunit GluA1 is lower in the araC condition than in insert, as
indicated by smaller GluA1 puncta in the araC group, there is no difference between
control and insert (Figure 3b).
• The number of synapses also seems to be similar between the control and insert
conditions (Figure 3a).
• We are currently working on evaluating the distance between synapses and the
soma in the three groups.
• A potential explanation for the discrepancy between the electrophysiology and the
immunocytochemistry data has to do with inhibition of synaptic transmission by
adenosine released from glia2. In the control cultures, where glia are present, synaptic
transmission might be inhibited at synapses contacted by glia. Since the insert has
been treated with araC, there are no glia present to release adenosine. Future
directions involve recording mEPSCs in presence of adenosine analogs.
Conclusion
a
Electrophysiology and Data Analysis:
• Whole-cell patch clamping was used to record miniature excitatory postsynaptic
currents (mESPCs) from pyramidal cells at 20-24 days in vitro.
• For each recording, a mean mEPSC amplitude and a mean inter-event interval was
calculated and averaged for the different treatment groups and plotted in a bar
graph. Student’s t-test was used for analysis.
Immunocytochemistry and Data Analysis:
• 20-24 days in vitro neurons were stained for GluA1, Synapsin, MAP2 and Hoechst.
• Puncta colocalization and average puncta size for GluA1 and Synapsin was
calculated and plotted in a bar graph. One-way ANOVA was used for statistical
analysis.
• p value smaller than 0.05 is denoted by "*" in the figures. Throughout, error bars
represent standard error of the mean.
b
c
• Pure neuronal cultures, grown in the presence of a cell culture insert
containing neurons and glia, are healthy and have well functioning synaptic
transmission.
• The insert restores the number of synapses and pre- and postsynaptic
components to control levels.
References
1.
2.
Figure 3: The insert rescues the number of synapses to control levels , increases the
number of AMPA receptor subunit GluA1 and decreases Synapsin to control levels. Synapses
were defined as areas where GluA1 and synapsin puncta co-localized. Puncta size for GluA1 and synapsin was taken to reflect the number of
molecules. (a) shows the number of synapses per μm2 of neuron (as measured by MAP2 positive area). The control group, on average, had about the
same number of synapses as the insert group, which was significantly higher than the araC group. (b) shows the average size of GluA1 positive
puncta. The size of puncta in control was similar to the insert. However, GluA1 puncta in the araC group were significantly smaller than in the insert
group. (c) shows the average size of synapsin puncta. Puncta were significantly larger in the araC condition than in control and insert. The
experiments for (a) and (b) were repeated twice and each condition had about 15 images taken from one experimental set. The resulting number of
images (n) per condition was as follows: control (n= 41), araC (n= 46), and insert (n= 37).
3.
4.
5.
Allen and Barres (2009) “Glia-more than just brain glue.” Nature, 457: 675-677.
Dunwiddie and Masino (2001) "The role and regulation of adenosine in the central nervous system." Annual Review of
Neuroscience, 24: 31-55.
Paixao and Klein (2010) “Neuron-astrocyte communication and synaptic plasticity.” Current Opinion in Neurobiology, 20: 466-473.
Paolicelli et al. (2011) “Synaptic Pruning by Microglia is Necessary for Normal Brain Development.” Science, 333: 1456-1458.
Ullian et al. (2001)“Control of Synapse Number by Glia.” Science, 291: 657-660.
Acknowledgments
I thank Dr. Kelsey Martin for her exceptional support and encouragement, Dr. Klara Olofsdotter for being an
incredible and supportive mentor and for teaching me electrophysiology, Victoria Ho for providing cells to use, Dr.
Besim Uzgil for assisting with electrophysiology analysis, Patrick Chen for assisting with immunocytochemistry
analysis, the Martin lab for their support, Dr. Roman Ferede for encouraging my interest in research, Dr. Richard
Russell for inspiring me to study neuroscience, Dr. Ira Clark for his support, and the Biomedical Research Summer
Scholarship, provided by the Minor in Biomedical Research, for funding this project.