1. Quantitative Comparison of Nuclear and Somatic
CaMKIV Expression in PyramidalVisual Cortex Neurons
Victor M. Suarez1, Anne Joseph2, Nathaniel Miska2, and Dr. Gina Turrigiano2*
1New Jersey Center for Science, Technology, and Mathematics Department, Kean University
2Department of Biology, Brandeis University
Abstract
Experimental Design
ConclusionData
Introduction
Future Works
Acknowledgements
Homeostatic plasticity is vital for neuron networks to maintain equilibrium in the face of
perturbations. This is accomplished through a series of mechanisms that allow neurons
to regulate their firing rates around a stable firing rate set point (FRSP). While this
phenomenon is observed both in vivo and in vitro, the process of maintaining a FRSP
is not very well understood. Calcium-calmodulin kinase IV (CaMKIV) is suspected to
play a critical role in the structure of FRSPs. We hypothesize that a drastic shift in
activity beyond the current FRSP results in CaMKIV activation leading to translocation
to the nucleus to regulate transcription. To test this, immunocytochemistry was utilized
to assess nuclear versus somatic CaMKIV expression and obtain a ratio of the two in
vitro in visual cortical pyramidal neurons treated with tetrodoxin (TTX) or bicuculline
(BCC). Preliminary data indicates that TTX treated cells exhibit significantly higher
nuclear/somatic CaMKIV localization when compared to control cells; however, BCC
data indicates no significant difference. Results suggest an inverse relationship
between CaMKIV and activity to moderate neuron homeostasis.
CaMKIV is a member of the Ca2+/calmodulin-
dependent protein kinase family which is
activated by an influx of intracellular calcium
ions causing translocation from the soma to
the nucleus of the cell. Once in the nucleus,
CaMKIV is known to be involved in the
phosphorylation of transcription factors and
regulation of genes critically involved in
synaptic and intrinsic excitability. TTX, a
potent neurotoxin that causes drastic
depression in cell firing rate activity, and
BCC, a competitive antagonist of GABA
receptors that induces an epileptic increase
of firing rate activity, were used to observe
cytoplasmic vs nuclear CaMKIV expression
under two extreme perturbations to attempt
to elucidate what role CaMKIV plays in
homeostatic regulation.
• I would like to extend my thanks to Dr. Gina Turrigiano for
allowing me to work in her lab, to my mentors Anne Joseph
and Nathaniel Miska for providing guidance, and to Darred
Surin and Kamil Moroz for their support.
• This project was made possible through the generous funding
and support of the Cell and Molecular Visualization REU
Grant REU DBI 1359172.
• Additional funding provided by the Garden State Louise
Stokes Alliance for Minority Participation (GS-LSAMP) Kean
University Chapter.
References
• Joseph, A. & Turrigiano G.G.
• Ibata, K., Sun, Q., & Turrigiano, G. G. (2008). Rapid synaptic scaling induced by
changes in postsynaptic firing. Neuron, 57(6), 819-826.
• Soderling, T. R. (1999). The Ca 2+–calmodulin-dependent protein kinase cascade.
Trends in biochemical sciences, 24(6), 232-236.
• Turrigiano, G. G. (2008). The self-tuning neuron: synaptic scaling of excitatory
synapses. Cell, 135(3), 422-435.
• Wayman, G. A., Lee, Y. S., Tokumitsu, H., Silva, A., & Soderling, T. R. (2008).
Calmodulin-kinases: modulators of neuronal development and plasticity. Neuron, 59(6),
914-931.
• Immunofluorescence Background [Online image]. (2016).Retrieved July 16, 2016 from
http://www.di.uq.edu.au/sparqcbeifbackground
A
Figure 1A: CaMKIV pathway cascade
resulting in scaling due to unknown scaling
factor. (Turrigiano et al 2008)
Figure 1B: Simplified CaMKIV pathway
depicting translocation from the soma to the
nucleus activating transcription (Joseph et
al, adapted from Wayman et al 2008.
A B
B
C
Quantification of Fluorescence Intensity
Figure 2A: Experimental workflow where each arrow represents an individual step preformed. Experiments typical ran 5 to 6 days per dissociation.
Figure 2B: Neurons were transfected with GFP, treated with BCC/TTX for six hours, and then mounted with nuclear DAPI stain (DIV8).
Figure 3B: Neurons imaged and average fluorescence intensity was measured and quantified using MetaMorph software. Data suggests that
nuclear CaMKIV expression increases in the TTX condition implying that transcription factors have been activated in response to the decrease of cell
activity. In contrast, nuclear CaMKIV expression in BCC treated cells resulted in highly variable data.
A
B
C
Figure 2: Representative images of selected neurons using LAS imaging software. GFP (green) was utilized to identify neurons on classical pyramidal morphology. CaMKIV (red) expression was measured and DAPI (blue) was used to
identify nuclear CaMKIV. Figure 2A: Stained control cells demonstrated a high variability in CaMKIV expression in transfect vs non-transfected neurons. Figure 2B: TTX treated cells expressed higher levels of CaMKIV in the nucleus
implying that transcription factors have been activated in response to the decrease of cell activity. Figure 2C: BCC treated cells expressed no clear trend in CaMKIV expression resulting in highly variable data.
Figure 3A: BCC treated cells produced results that were opposite of the original hypothesis. Nuclear
CaMKIV expression appears more intense when compared to somatic expression implying that
CaMKIV is still being activated. While a trend of nuclear CaMKIV expression is seen when compared
to control cells, no significant conclusions can be drawn at this time.
Figure 3B: TTX treated cell data supported the original hypothesis that when cells respond to a decrease in
activity by increasing CaMKIV in attempt to scale up activity to a new set point through transcription factor
activation. The average nuclear CaMKIV (*) expression (p=0.0644) is close to being significant.
Figure 3C: Non-transfected pyramidal cells were
imaged to investigate the high variability in CaMKIV
expression when compared to transfected cells. The
average nuclear to somatic CaMKIV (**) expression
ratio (p=0.0065) showed approximately a two-fold
increase. Nuclear CaMKIV (***) expression
(p=0.0494) showed an approximate four-fold
increase when compared to control cells.
There is no clear reason as to why the fluorescence
intensity increases so dramatically in the non-
transfected cells. It is unknown if BCC treated, non-
transfected cells would express a similar trend.
• Non-transfected cells treated with TTX showed a
significant increase in nuclear CaMKIV relative to
somatic CaMKIV.
• Cells transfected with GFP showed a nonsignificant
trend toward increased nuclear/somatic CaMKIV when
treated with either TTX or BCC.
• Non-transfected neurons appear to express more
CaMKIV when compared to transfected neurons.
• TTX-treated cells demonstrate an inverse ratio of
CaMKIV to activity (i.e. low activity, higher CaMKIV and
vice versa).
• BCC-treated cells elicit nuclear CaMKIV localization
similar to TTX-treated cells, paradoxically.
• Investigate why non-transfected cells express such a
significant increase in CaMKIV expression when
compared to transfected cells in the TTX condition.
• Determine if non-transfected cells treated with BCC
demonstrate similar changes in expression when
compared to transfected cells.
• Implement a more reliable method of pyramidal cell
identification than only morphology such as NeuN stain.
• Run treatments for 2, 4, and 6 hour intervals to
determine is there is ceiling/floor level of activity.
A
*
B
**
***
C