Controlling anoxic tolerance in adult Drosophila via the cGMP-PKG pathway.

In this study we identify a cGMP-dependent protein kinase (PKG) cascade as a biochemical pathway critical for controlling low-oxygen tolerance in the adult fruit fly, Drosophila melanogaster. Even though adult Drosophila can survive in 0% oxygen (anoxia) environments for hours, air with less than 2% oxygen rapidly induces locomotory failure resulting in an anoxic coma. We use natural genetic variation and an induced mutation in the foraging (for) gene, which encodes a Drosophila PKG, to demonstrate that the onset of anoxic coma is correlated with PKG activity. Flies that have lower PKG activity demonstrate a significant increase in time to the onset of anoxic coma. Further, in vivo pharmacological manipulations reveal that reducing either PKG or protein phosphatase 2A (PP2A) activity increases tolerance of behavior to acute hypoxic conditions. Alternatively, PKG activation and phosphodiesterase (PDE5/6) inhibition significantly reduce the time to the onset of anoxic coma. By manipulating these targets in paired combinations, we characterized a specific PKG cascade, with upstream and downstream components. Further, using genetic variants of PKG expression/activity subjected to chronic anoxia over 6 h, approximately 50% of animals with higher PKG activity survive, while only approximately 25% of those with lower PKG activity survive after a 24 h recovery. Therefore, in this report we describe the PKG pathway and the differential protection of function vs survival in a critically low oxygen environment.

Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo.

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila.

Previous studies suggest that the vesicular cysteine-string protein (CSP) may modulate presynaptic Ca(2+) channel activity in fast neurotransmitter release. To test this hypothesis, we analyzed the dynamics of presynaptic Ca(2+) ion influx with the Ca(2+) indicator fluo-4 AM at csp mutant neuromuscular junctions of Drosophila. From 24 to 30 degrees C, stimulus-evoked, relative presynaptic Ca(2+) signals were increasingly larger in csp mutant boutons than in controls. Above 30 degrees C, Ca(2+) signals declined and were similar to controls at 34 degrees C. A prolonged decay of Ca(2+) signals in mutant boutons at high temperatures indicated abnormally slow Ca(2+) clearance. Cytosolic Ca(2+) at rest was determined with the ratiometric Ca(2+) indicator fura-2 AM and was similar in mutant and control boutons at 24 degrees C but higher in mutant boutons at 34 degrees C. Despite larger Ca(2+) signals in mutant boutons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca(2+) entry cannot explain the reduction of neurotransmitter release in csp mutants. At all temperatures tested, raising extracellular Ca(2+) increased transmitter release elicited by single stimuli in csp mutants. Collectively, these data suggest multiple functions for CSP at synaptic terminals. Increased Ca(2+) signals coupled with reduced release suggest a direct function of CSP in exocytosis downstream from Ca(2+) entry. Because the reduction of evoked release in csp mutants is counteracted by increased Ca(2+) levels, we suggest that CSP primarily increases the Ca(2+) sensitivity of the exocytotic machinery.

Heat shock protects synaptic transmission in flight motor circuitry of locusts.

We investigated the effects of heat shock on the temperature sensitivity of synaptic transmission in the motor circuit for flight in Locusta migratoria. In heat shocked animals synaptic transmission failed at 5-6 degrees C higher than in control animals and recovery of transmission was more than three times faster upon return to room temperature. We also found that synaptic delay was rendered insensitive to increases in temperature by heat shock. Thus we have shown in the locust that heat shock has important protective effects on synaptic transmission, thereby extending the upper temperature limit for the motor patterns that generate flight. This is the first description of an effect of heat shock that preserves neuronal communication under subsequent stressful conditions.

Forewing asymmetries during auditory avoidance in flying locusts

Flying locusts orient to sounds in their environment. Sounds similar to those produced by echolocating bats cause a flying locust to change its flight path. We used high-speed cinematography and videography to study changes in body posture and wing kinematics of tethered locusts in response to stimulation with bat-like sounds. Locusts showed both negative and positive phonotaxis to this stimulus. Within a few wingbeats of stimulus onset (between 126 and 226ms), locusts deflected their abdomens to one side, and the angle of the left and right forewings with respect to the dorsal­ventral body axis became asymmetrical during the downstroke. This forewing asymmetry, in which the forewing on the inside of the turn became more depressed, ranged from 20 to 45° (37±9.7°, mean ± s.d.) and was correlated with the direction and magnitude of abdomen deflection, a measure of steering in tethered, flying locusts. Hindwing stroke angle asymmetries were minimal or non-existent after stimulation. Coincident with changes in forewing asymmetry and abdomen deflection was a decrease in stroke amplitude (19±6.5°) of the forewing on the inside of the attempted turn. Motor patterns from forewing first basalar (M97) muscles showed an asymmetry in the timing of left and right depressor activation that ranged from 10.4 to 1.6ms (4.23±2.85ms). The number of spikes per depressor burst increased to a maximum of three spikes in the muscle on the inside of the attempted turn, and depressor frequency (wingbeat frequency) increased by approximately 2Hz (2.17±0.26Hz). We suggest that the asymmetry in forewing first basalar activity is causally related to the asymmetry in the timing of the initiation of the downstroke, resulting in an asymmetry in the ranges of the stroke angles of the forewings, which would impart a roll torque to the locust. This would augment the steering torques generated by concurrent changes in the angle of attack of the fore- and hindwings and changes in abdomen position to effect rapid avoidance manoeuvres.

Exposure to heat shock affects thermosensitivity of the locust flight system.

The natural habitat of the migratory locust, Locusta migratoria, is likely to result in locusts being heat stressed during their normal adult life. It is known that locusts exhibit a heat-shock response: exposure to 45 degrees C for 3 h induces thermotolerance and the expression of heat-shock proteins. We investigated the effects of exposure to heat-shock conditions on the thermosensitivity of flight rhythm generation in tethered, intact animals and in deafferented preparations. Heat shock had no effect on wingbeat frequency measured at the start of flight sequences, nor did it affect the postimaginal maturation of this parameter. During sustained flight, heat shock slowed the characteristic asymptotic reduction of wingbeat frequency. Wingbeat frequency of heat-shocked animals was less sensitive to temperature in the range 24 degrees to 47 degrees C than that of control animals, and the upper temperature limit, above which flight rhythms could not be produced, was 6 degrees to 7 degrees C higher in heat-shocked animals. These results were mirrored in the response of deafferented preparations, indicating that modifications in the properties of the flight neuromuscular system were involved in mediating the response of the intact animal. We propose that exposure to heat shock had the adaptive consequences of reducing thermosensitivity of the neural circuits in the flight system and allowing them to operate at higher temperatures.