Hu Z , Du Y , Xiao X , Dong K , Wu W .

GM 080255 NIGMS NIH HHS , R01 GM080255 NIGMS NIH HHS , R01 GM057440 NIGMS NIH HHS

	Figure 1. HA delayed the decay rate of the evoked EJPs at the Drosophila neuromuscular junction. (A) representative EJPs traces in control and in the presence of HA (2.5 × 10−6 M). (B) mean amplitude of evoked EJPs before and after 3 min of HA application. Evoked EJPs were elicited by stimulating the segmental nerve at 0.1 Hz with the amplitude of the pulse of 5V. The values reported are the mean ± SD. (C) mean amplitude of evoked EJPs at various time points (3, 10, 20, 40, and 60 min) in control and in the presence of HA were normalized to the initial amplitude of evoked EJPs, i.e., n = 7 for the control and n = 14 for HA treated. Statistical significance was determined by Student’s t-test, and significant values were set at * p < 0.05 and ** p < 0.01.
	Figure 2. HA increased the frequency of mEJPs at the Drosophila neuromuscular junction. (A) I, representative of mEJP traces in the control and in the presence of HA (2.5 × 10−6 M). II, overlap and summation of mEJPs recorded 20 min after HA application, which occurred in about 33% of total recordings. Arrows indicate the irregular mEJPs. (B) analysis of mEJP amplitudes; HA did not affect the average amplitude of mEJPs. I, box plots of mEJPs amplitudes. II, mEJPs amplitude before and at 3 min after HA application showing no significant difference; III, mean mEJPs amplitude at different time points (3, 10, 20, 40, and 60 min) in the control and HA application were normalized to the initial mEJPs amplitude in the control and HA application, separately. No significant difference was observed with time on mEJPs amplitude in both control and HA application (p > 0.05). (C) analysis of mJPs frequencies, HA induced a significant enhancement in the frequency of mEJPs. I, mEJP frequency before and after 3 min of HA application; there is no significant difference (p > 0.05); II, mEJP frequency at different time points (3, 10, 20, 40, and 60 min) in the control and HA application were normalized to the initial mEJP frequency in the control and HA application, separately. mEJP frequency in the control showing significant decrease with time, whereas HA induced a significant increase at 40 and 60 min. Notably, the start and end values of mEJPs amplitude/or frequency in (C) are the EJP amplitude/or frequency at 3 min after application and other different recording times, respectively. The values reported in (C) are the mean ± SD. HA, n = 7; control n = 6. Statistical significance was determined by Student’s t-test, and significant values were set at * p < 0.05 and ** p < 0.01, *** p < 0.001.
	Figure 3. HA induced a significant hyperpolarization direction shift in inactivation-gating properties in cockroach sodium channels BgNav1-1a. (A) recording traces before and after the application of 10−5 M of HA; (B) I-V curve of cockroach sodium channels BgNav1-1a before and after the application of 10−5 M of HA; (C) and (D) the voltage dependence of activation (C) and inactivation (D) before and after the application of 10−5 M of HA. The activation and inactivation curves were fitted with two-state Boltzmann equations. The number of oocytes was 5–8; (E) and (F) I-V curve of Drosophila sodium channels DmNav22 (E) and mosquito sodium channels AaNav1-1 (F) before and after the application of 10−5 M of HA.
	Figure 4. Structure of haedoxan A.

Boudier, Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes. 1992, Pubmed

Boudier, Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes. 1992, Pubmed
Catterall, From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. 2000, Pubmed
Choi, Miniature neurotransmission regulates Drosophila synaptic structural maturation. 2014, Pubmed
Collins, Synaptic development: insights from Drosophila. 2007, Pubmed
Du, Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides. 2009, Pubmed
Du, Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. 2013, Pubmed , Xenbase
Flucher, Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kd protein. 1989, Pubmed
Krans, The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration. 2010, Pubmed
Lee, Brine shrimp lethality of the compounds from Phryma leptostachya L. 2002, Pubmed
Lupa, Aggregation of sodium channels during development and maturation of the neuromuscular junction. 1993, Pubmed
Mahmud, Na+ channels at postsynaptic muscle membrane affects synaptic transmission at neuromuscular junction: a simulation study. 2012, Pubmed
Motomura, Temperature dependence of pyrethroid modification of single sodium channels in rat hippocampal neurons. 2000, Pubmed
Olson, Molecular and functional characterization of voltage-gated sodium channel variants from Drosophila melanogaster. 2008, Pubmed , Xenbase
Ormerod, Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae. 2013, Pubmed
Park, Larvicidal activity of lignans identified in Phryma leptostachya Var. asiatica roots against three mosquito species. 2005, Pubmed
Peled, Evoked and spontaneous transmission favored by distinct sets of synapses. 2014, Pubmed
Pugsley, Spiradoline, a kappa opioid receptor agonist, produces tonic- and use-dependent block of sodium channels expressed in Xenopus oocytes. 2000, Pubmed , Xenbase
Ramirez, Differential regulation of spontaneous and evoked neurotransmitter release at central synapses. 2011, Pubmed
Rees, The effect of metabolic inhibitors on the cockroach nerve-muscle synapse. 1974, Pubmed
Seo, Larvicidal activity of medicinal plant extracts and lignan identified in Phryma leptostachya var. asiatica roots against housefly (Musca domestica L.). 2012, Pubmed
Stewart, Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. 1994, Pubmed
Stewart, Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology. 1996, Pubmed
Stimson, A product of the Drosophila stoned locus regulates neurotransmitter release. 1998, Pubmed
Tan, Identification of amino acid residues in the insect sodium channel critical for pyrethroid binding. 2005, Pubmed , Xenbase
Tan, Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. 2002, Pubmed , Xenbase
Ulbricht, Sodium channel inactivation: molecular determinants and modulation. 2005, Pubmed
Xiao, Larvicidal activity of lignans from Phryma leptostachya L. against Culex pipiens pallens. 2012, Pubmed
Xiao, Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels. 2001, Pubmed
Yamauchi, Synthesis and Insecticidal Activity of Lignan Analogs (II). 1992, Pubmed