NMDA receptors on zebrafish Mauthner cells require CaMKII‐α for normal development

Abstract

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

Zebrafish TARP Cacng2 is required for the expression and normal development of AMPA receptors at excitatory synapses

“…The morphants show evidence of curved tails, cardiac edema and larger yolk sacs, while con-trol injected embryos appear to be morphologically and developmentally normal and are similar to uninjected clutch mates. Morphants experienced about 85-90% knockdown of targeted mRNA, as determined by quantification of cDNA copy number following quantitative real time PCR (Roy et al, 2014). We determined the efficacy of the morpholinos in this manner rather than by examining PCR gels for exon skipping because the Cacng2a morpholino is designed against an overlapping region between exon 1 and intron 1, which is 84 kB in size.…”

“…Zebrafish from the Oregon AB wild-type line were obtained from the University of Oregon and raised in the aquatics facility in the Department of Biological Sciences at the University of Alberta according to established protocols (Patten and Ali, 2007;Patten and Ali, 2009;Roy et al, 2014). Embryos were raised at 28.58C in Egg Water (60 lg mL 21 "Instant Ocean," United Pet Group, Cincinnati, OH) or Embryo Medium (mM: 13.7 NaCl; 0.54 KCl; 0.025 Na 2 HPO 4 ; 0.044 KH 2 PO 4 ; 1.3 CaCl 2 ; 1.0 MgSO 4 ; 0.42 NaHCO 3 ; pH 5 7.2) containing Methylene Blue (Sigma, St. Louis, MO; 0.003%) and PenStrep (Sigma; 10 mL L 21 ) until required for experiments.…”

Gjd2b-mediated gap junctions promote glutamatergic synapse formation and dendritic elaboration in Purkinje neurons

“…Total RNA (2ng/µl) from such replicates was used for cDNA synthesis using Superscript III reverse transcription kit according to manufacturer’s protocol (Invitrogen, USA). Absolute copy numbers were obtained by droplet digital PCR (ddPCR) using Evagreen mastermix (Biorad, USA) and gene-specific primers that were previously described for zebrafish genes[66] ( camk2a, camk2b1, camk2b2, camk2d1, camk2d2, camk2g1, camk2g2 , and a reference control ef1 α). Absolute quantification in the form of number of copies per μL for each camk2 isoform was normalized with the control ef1 α copy numbers in each replicate.…”

Cucurbitacin IIa: A review of phytochemistry and pharmacology

“…Studies have shown that the expression of BDNF is regulated by CaMKII α/CREB/BDNF pathway (Tang et al, 2019). Calcium/ calmodulin-dependent protein kinase 2 (CaMKII) is a protein associated with synaptogenesis and neural development, which exists in synapses and is rich in content (Roy, Ferdous, & Ali, 2015). The transcription factor cAMP response element-binding protein (CREB) is involved in the memory process and is also an important target for the treatment of depression (Carlezon Jr., Duman, & Nestler, 2005).…”