Locomotor performance and CNS responses to hypoxia in a cyclic nucleotide-gated channel mutant of adult Drosophila

Drosophila provides an excellent opportunity to explore the genetic basis for behavioral and CNS responses to hypoxia. Cyclic guanosine monophosphate (cGMP) modulates the speed of recovery from anoxia in adults and mediates hypoxia-related behaviors in larvae. Cyclic nucleotide-gated channels (CNG) and cGMP-activated protein kinase (PKG) are two cGMP downstream targets. PKG is involved in behavioral tolerance to hypoxia and anoxia in adults, however little is known about CNG channels. We used a CNGL mutant with reduced CNGL transcripts to investigate the contribution of CNGL to the hypoxia response. In control flies (w1118), hypoxia immediately reduced path length per minute in a locomotor assay. Flies took 30-40 mins in air to recover from 15 mins hypoxia. CNGL mutants had reduced locomotion under normoxia and impaired recovery from hypoxia, similar to the effects of pan-neural CNGL knockdown. In the CNGL mutants hypoxia caused an acute increase in path length per minute followed by a gradual increase during hypoxia. Basal levels of CNS extracellular K+ concentrations were reduced in the mutants. In response to hypoxia, the mutants had an increased extracellular K+ concentration change, reduced time to reach the K+ concentration peak, and delayed recovery time. Genetic manipulation to increase cGMP in the CNGL mutants eliminated the impairment of recovery from hypoxia and partially compensated for the effects of hypoxia on CNS K+. Although the neural mechanisms have yet to be determined, CNGL channels and cGMP signaling are involved in the hypoxia response of adult Drosophila.

(1) Contribution of CNGL to the hypoxia response was investigated in Drosophila adults.

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(2) CNGL mutants with low CNGL transcripts showed reduced locomotion in normoxia 18 and poor recovery from hypoxia.

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(3) CNGL knockdown in neurons replicated the properties of the CNGL mutants.  3) A CNGL homolog in Hawaiian crickets is a candidate gene underlying interspecific 103 variation in centrally-generated song patterns, which contribute to the rapid evolution of 104 reproductive barriers and speciation (74).

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Considering that cGMP and PKG are involved in responses to hypoxia and that CNG 106 channels contribute to the escape response to hypoxia in Drosophila larvae yet CNGL 107 channels do not, we investigated the role of the CNGL channel in regulating the hypoxia 108 response of adult flies. We hypothesized that mutation of CNGL would alter behavioral and 109 electrophysiological responses to hypoxia and tested the hypothesis using a locomotor 110 assay and comparing CNGL mutant flies and w1118 control flies under normoxia, under 111 hypoxia, and during recovery. In addition, we examined the effects of hypoxia on 112 locomotion in pan-neural or pan-glial CNGL knockdown flies. We measured extracellular 113 K + concentration in the brain before and during hypoxia to investigate possible changes 114 specific to K + rather than overall ion homeostasis. In addition, we used fly lines with 115 overexpression of Gyc88E or mutation of Pde1c to examine the interaction between CNGL 116 and cGMP in response to hypoxia.

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Male flies were collected within two days after eclosion and raised for at least three 144 additional days before the experiments. 100% N2 gas was used to knock down flies during 145 collection but flies were not exposed to 100% N2 for 3 days prior to testing. All 146 experiments took place between 10 am and 4 pm on flies younger than 9 days old.    Figure   479 2D-6D)). The peak value of this acute increase in path length per minute was then 480 compared with that at 10 min, which was the last minute before hypoxia. In most cases, the 481 peak value was significantly higher than that at 10 min ( Figure 6A Figure 7E, the value was not remarkably higher than that 486 at 10 min, which might be due to large variations (P > 0.05, Mann-Whitney test). The current study has shown that the overexpressing Gyc88E compensated for the 571 reduced locomotion in CNGL mutant after hypoxia. We then tested whether it could also 572 eliminate the disruption to ion homeostasis in response to hypoxia in CNGL mutant flies.

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In summary, our results support the conclusion that the CNGL channel is involved in 772 ion homeostasis in the brain and the recovery from hypoxia in Drosophila adults. With 773 respect to central pattern generation, the role of cGMP and CNGL channels in episodic 774