ABSTRACT
A key step in the maturation of glutamate synapses is the developmental speeding of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPA-R) kinetics, which occurs via a switch in receptor subtypes. However, the molecular components required for the switch in receptors are unknown. Here, we used the zebrafish preparation to show that activation of protein kinase C (PKC)gamma is necessary for the developmental speeding of AMPA-R kinetics. Targeted knockdown of PKCgamma with an antisense morpholino oligonucleotide [PKCgamma-morpholino (PKCgamma-MO)], prevents the normal speeding up of AMPA-R kinetics in Mauthner cells. PKCgamma-MO-injected embryos are incapable of trafficking AMPA-Rs following application of phorbol 12-myristate 13-acetate or PKCgamma. PKCgamma-MO-injected embryos do not hatch or exhibit the C-start escape response. Increasing synaptic activity (33 h post-fertilization embryos) by application of an elevated K(+) medium or by application of N-methyl-D-aspartate induces rapid PKCgamma-dependent trafficking of fast AMPA-Rs to synapses. Our findings reveal that PKCgamma is a molecular link underlying the developmental speeding of AMPA-Rs in zebrafish Mauthner cells.
Subject(s)
Neurons/physiology , Protein Kinase C/metabolism , Receptors, AMPA/metabolism , Rhombencephalon/embryology , Rhombencephalon/physiology , Synapses/physiology , Animals , Biological Transport, Active/drug effects , Biological Transport, Active/physiology , Central Nervous System Agents/pharmacology , Embryo, Nonmammalian/physiology , Excitatory Postsynaptic Potentials , Gene Knockdown Techniques , Immunohistochemistry , Kinetics , N-Methylaspartate/metabolism , Neurons/drug effects , Patch-Clamp Techniques , Potassium/metabolism , Protein Kinase C/genetics , Rhombencephalon/drug effects , Signal Transduction , Synapses/drug effects , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , Zebrafish/embryologyABSTRACT
In some cells, the development of voltage-gated channels requires synaptic input, while in others it does not. Here we investigate whether the sodium and potassium currents in the skeletal muscle of zebrafish sofa potato (sop(-/-)) mutants develop normally. Zebrafish sop(-/-) mutants do not express nicotinic acetylcholine receptors at neuromuscular junctions, and therefore do not exhibit synaptic activity in muscle. We find that in both red and white muscle fibers, sop(-/-) mutants are able to support normal potassium current development during early stages of development [1-3 days post fertilization (dpf)], but at 6 dpf the potassium current density is significantly smaller than that in their phenotypically wild-type siblings (sop(+/?)). In contrast, sodium current density is unaffected. The steady-state properties of potassium currents are unaltered in the sop(-/-) mutants, but there is a significant difference in the V(50) of inactivation of sodium currents. This is the first study in zebrafish to investigate activity-dependent mechanisms of ion channel development and our results indicate that some aspects of ion current development in skeletal muscle require synaptic activity, whereas others do not.
Subject(s)
Muscle, Skeletal/physiology , Potassium Channels/physiology , Receptors, Nicotinic/deficiency , Sodium Channels/physiology , Zebrafish/physiology , Animals , Embryo, Nonmammalian , Membrane Potentials/physiology , Mutation , Patch-Clamp Techniques , Receptors, Nicotinic/genetics , Zebrafish Proteins/geneticsABSTRACT
Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor-related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non-mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12-h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles, smaller tail velocities and aberrant C-bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non-stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non-functional with respect to the development of AMPA synaptic transmission.