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

Abstract

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.

Dietary choline intake is necessary to prevent systems‐wide organ pathology and reduce Alzheimer's disease hallmarks

“…Interestingly Nptn, a critical protein for long‐term potentiation at Hp excitatory synapses, also linked to AD (Ilic et al, 2019 ), was downregulated in 3xTg‐AD ChN mice compared to NonTg ChN counterparts (Log 2 FC = −1.55), and Ch‐ downregulated this protein in both NonTg (Log2 FC = −2.13) and 3xTg‐AD mice (Log 2 FC = −3.44). Moreover, proteins that modulate AMPA receptors Cacng8 (Log 2 FC = −1.00), Lrrtm1 (Log 2 FC = −1.34), Frrs1l (Log 2 FC = −1.38), as well as NMDA receptors Grin2a (Log 2 FC = −1.49), Clstn1 (Log 2 FC = −1.05), were also downregulated by Ch‐ (Bhouri et al, 2018 ; Roy et al, 2016 ; Stewart et al, 2019 ), suggesting that Ch‐ can contribute to postsynaptic membrane dysfunction in AD. Previous work has shown that choline modulates the expression of alpha7 nicotinic acetylcholine and Sigma‐1 receptors (Velazquez et al, 2019 ).…”

Expression and Development of TARP γ-4 in Embryonic Zebrafish

“…In a previous study, we found that knockdown of zebrafish Stargazin γ-2a resulted in a reduction of AMPA mEPSC amplitude and frequency associated with Mauthner cells, and that the expression of AMPARs appeared to be reduced in embryonic brain [19]. To determine if knockdown of γ-4a or γ-4b had similar developmental effects on AMPA currents, we patch-clamp-recorded AMPA mEPSCs from M-cells of newly hatched embryos.…”

“…While TARPs have been extensively studied in mammals, there is very little known about their roles in nonmammalian vertebrates such as zebrafish. Our group was the first to present functional data on the role of the prototypical TARP γ-2, in developing zebrafish, and found it to be necessary for the trafficking of AMPAR to synaptic membranes [19], consistent with its roles in mammals. Few studies have focused on the role of other members of the type I family such as γ-4 or γ-8 in nonmammalian vertebrates.…”

Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

“…; Henley and Wilkinson ) and changes in escape responses (smaller tail velocities and aberrant turning directions) (Roy et al . ). It has been demonstrated that AMPARs mediate synaptic transmission from cone photoreceptors to OFF bipolar cells in zebrafish (Wong et al .…”