Differential expression of nicotinic acetylcholine receptor subunits is critical for the embryonic development of zebrafish muscle and neuromuscular junction

• Author / Creator

  Ahmed, Kazi Tanveer

• Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels primarily expressed on muscle fibers and neurons. They are comprised of five subunits arranged in different stoichiometries. nAChRs expressed at the neurons are comprised of different combinations of α1-10 and β1-4 subunits, whereas muscle acetylcholine receptors are comprised of α1, β1, δ and γ/ε subunits. At neuromuscular junctions (NMJs), nAChRs are activated by binding to the neurotransmitter- Acetylcholine (ACh), which is released from motor axon terminals. Activation of the receptors allows the passage of Na+, K+ and Ca2+ which leads to muscle depolarization. Thus, nAChRs play a critical role in muscle contraction and movement. Mammalian twitch muscle fibers express γ subunit containing receptors during embryonic stages, but this subunit is eventually replaced by ε subunit-containing receptors. Here, I studied zebrafish tonic red and twitch white muscle fibers to determine if the NMJs of zebrafish red and white fibers show a development pattern similar to mammalian muscle. Additionally, the scientific literature describing the development of zebrafish NMJs was inconsistent and controversial, and I sought to add clarity to the literature. My results show that the NMJs of zebrafish red and white muscle fibers show different synaptic properties during embryonic development. To determine the properties of nAChRs in developing fish I recorded mEPCs obtained from muscle fibers of animals aged between 1 dpf (day post fertilization) and 5 dpf and examined the characteristics of the mEPCs. I found that miniature end plate currents (mEPCs) recorded from white fibers exhibit single exponential decay at 1.5 dpf. However, by 2 dpf, mEPCs exhibit double exponential decays, and then revert to single exponential decays between 4 and 5 dpf. Therefore, by 5 dpf all mEPCs decay with fast, single exponential decays. In contrast, red fiber mEPCs exhibit double exponential decays throughout development between 1.5 and 5 dpf. Single channel recordings reveal the presence of long and short open channels with two conductance classes at 2 dpf in both fiber types. Red muscle fibers retain both the long and short open channels at 5 dpf whereas only short open channels are present in white muscle fibers at 5 dpf. RT-PCR of whole embryos shows that zebrafish express both the γ and ε subunits even in adult animals. But RT-qPCR from isolated red and white fibers indicate that ε subunits are predominantly expressed in white fibers by 5 dpf, whereas both γ and ε subunits are continuously express in red fibers. Next, to determine a role for the ε or γ subunits, I used morpholino oligonucleotides (MOs) to knockdown the expression of these subunits during early development. To achieve my goals, I used two sets of MOs. The first was a set of translation blocking MOs (MO-EM and MO-GM targeting ε and γ subunits respectively) previously used by Mongeon et al. (2011). I found that the development of motor neurons and muscle fibers, and locomotor activity of 2 dpf and 5 dpf animals were unaffected in the morphants. However, similar to published results, the decay kinetics of mEPCs obtained from 2 dpf white, 5 dpf red and 5 dpf white fibers were prolonged with MO-EM. These results suggested that the ε and γ subunits have little roles in zebrafish embryonic development. I observed that the concentration of the MOs injected by Mongeon et al. (2011) was nearly 1000 fold less than generally used concentrations. Therefore, I designed and injected a set of splice blocking MOs (MO-E and MO-G targeting ε and γ subunits respectively). RT-PCR confirmed that both splice-blocking MOs resulted in altered splicing. Zebrafish injected with MO-E exhibited slight tail curvature at both 2 dpf and 5 dpf, and reduced expression of nAChRs at 5 dpf. But similar to translation blocking MO, MO-E did not affect the development of motor neurons. In contrast, injection of MO-G resulted in severe morphological defects, and significantly affected the development of motor neuron branches. Expression of nAChRs was not greatly reduced but showed a disorganized pattern. Whole cell recordings of mEPCs showed properties that were mostly similar to those obtained from the translation blocking MOs, where MO-E resulted in increased decay times of mEPCs from white muscle fibers. Red muscle fibers remained unaffected. Synaptic properties of both red and white fibers remained unaffected following MO-G injections. The single channel properties of nAChRs were unaffected in MO-G morphants. Injection of MO-E resulted in a greater number of long open channel activity in red fibers, whereas single channel recordings from white fibers were rarely observed and were largely non-existent. The locomotor activity of 2 dpf embryos and 5 dpf larvae was also severely affected by MO-G, but not by MO-E. Therefore, the ε subunit appears to affect the synaptic properties of only white fibers and not red fibers. In contrast, the γ subunit appears to have minimal effects on synaptic properties but a much greater effect on motor neuron development and locomotion. Thus, my results show that the ε and γ subunits of nAChRs play different roles in embryonic development of zebrafish red and white muscle fibers.

• Subjects / Keywords

  • Tonic vs twtich muscle fibers
  • nAChR
  • Red and White muscle fiber
  • NMJ
  • Embryonic development
  • Zebrafish

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-znqa-z626

• License

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