â¢Travel assistance was provided by The Mary Louise Imrie Graduate Student Award. â¢Special thanks to Troy Locke of MBSU, Department of Biological Sciences ...
CaMKII is involved in the recruitment of AMPA receptors to excitatory synapses on zebrafish Mauthner neurons 540.13
B Roy*, C Collins & D W Ali, University of Alberta, Edmonton, Canada
INTRODUCTION
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CaMKII inhibitors have no effect on mEPSc frequency
•Calcium/calmodulin dependent protein kinase 2 (CaMKII) plays important roles in glutamate AMPA receptor trafficking and neuronal plasticity1.
33 hpf
6
48 hpf
Single Cell RT-qPCR: ds DNA standards generated unreliable results at lower dilutions.
•AMPARs mediate the majority of the fast excitatory synaptic transmission in zebrafish Mauthner neurons2. •We wanted to determine if CaMKII, which is expressed in developing zebrafish3, plays a role in the modulation of AMPA currents as well as in the normal development of AMPA receptor-containing synapses.
MATERIALS & METHODS
Amplification of 100, 10 and 2 template
Preparation - Embryos and newly hatched larvae were anaesthetized and the entire hindbrain was exposed. RB
M-cell VIII
Mn
Mcell
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Muscle
15 µm
KN-62 and AIP increase the mEPSC rise time in 48hpf fish but not in the 33hpf fish. 33 hpf
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Too little cDNA from single Mauthner cell resulted in non-reproducible amplification.
48 hpf
Electrophysiological Recording - We used whole cell patch-clamp electrophysiology to record from Mauthner neurons. The extracellular recording solution contained (in mM) 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, osmolarity adjusted to 290 mOsm, pH 7.8. A 10mM K+ saline medium was applied to depolarize the cells. The intracellular solution was composed of (in mM) 115 Cs-gluconate, 15 CsCl, 2 MgCl2, 10 HEPES, 10 EGTA, and 4 Na2ATP, osmolarity adjusted to 280290 mOsm, pH 7.2. To block CaMKII activity, either a general CaMK blocker KN-62 (10 µM) or a specific peptide inhibitor AIP (5 µM) was included in the intracellular solution. Western Blot – Zebrafish brains were dissected, pooled together and probed with pan anti-CaMKII primary antibody after homogenization. Single-Cell RT-qPCR – DNA fragments spanning the qPCR products for seven CaMKII genes were amplified from cDNA isolated from a pool of 30 fish at 48 hours post fertilization (hpf). After purification, these ds DNA fragments were used to construct a 10 fold serial dilution. Gene specific primer and probe sets were obtained from Roche.
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CaMKII inhibitors block the increase in AMPA mEPCs amplitude in 33 and 48 hpf fish. 33 hpf
CaMKII inhibitors increase the time constant in 48hpf fish. 33 hpf
48 hpf 1. CaMKII appears to modulate AMPA mEPSCs differentially during zebrafish development. 2. Since inhibition of CaMKII activity results in the reduction of mEPSC amplitude, but not frequency, our results suggest that CaMKII might be involved in AMPAR trafficking and/or anchoring to the PSD.
48 hpf Control
3. RT-qPCR from the Mauthner cell is an ongoing project. Although we met with various challenges, we think the expression profile of CaMKII genes in Mauthner cell would be invaluable for investigating the role of different CaMKII isoforms in zebrafish neuronal development.
Control + 10mM K+ 50pA 5pA 2 ms
CONCLUSIONS
0.5 s
REFERENCES (1) Lisman J et al. (2002) Nat Rev Neurosci 3:175-190. (2) Ali DW et al. (2000) J Neurophysiology. 83:181-191.
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Western blot confirms the presence of CaMKII in developing zebrafish.
75 KDa 50 KDa -
CaMKII
(3) Rothschild SC et al. (2007) Dev Dyn 236:295-305.
ACKNOWLEDGEMENTS •This research was funded by an operating grant (NSERC) and equipment grant (CFI) to DWA. •Travel assistance was provided by The Mary Louise Imrie Graduate Student Award. •Special thanks to Troy Locke of MBSU, Department of Biological Sciences for his continuous support for the qPCR analysis.
