Neuroprotection at Drosophila synapses conferred by prior heat shock

Abstract

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Here we show that synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock that strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, we found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties.

Fig. 1.

Transient expression of hsp70 inDrosophila larvae after heat shock. Whole wandering third-instar larvae were exposed to either a 1 or 2 hr heat shock at 36°C (a, b, respectively) and then placed at a recovery temperature of 25°C for the indicated number of hours. Whole-body lysates of the larvae were analyzed by Western blotting to detect hsp90, hsp70, and hsp60 (50 μg of protein loaded per lane). The recovery interval at 25°C following heat shock is indicated in hours. C, Control larvae raised at 25°C; HS, larvae exposed to heat shock at 36°C for either 1 or 2 hr.

Fig. 2.

Macropatch recordings from individual, visualized synaptic boutons of the larval neuromuscular junction. Placement of the focal macropatch electrode over individual Ib boutons at the larval neuromuscular junction was under visual control to record synaptic currents generated at that site. a, Strings of Ib and Is boutons can be seen innervating the surface of muscle 6 under Nomarski optics. b, Under DiOC₂(5) fluorescence, the same string of Ib boutons can be seen clearly, and the surrounding subsynaptic reticulum (SSR) does not fluoresce; however, fluorescence appears where the SSR meets the muscle. c, Overlay of the fluorescence image on the Nomarski image is shown.d, The focal macropatch electrode is gently placed over the chosen Ib bouton. The bouton can be seen through the lumen of the electrode. At this concentration of DiOC₂(5) and exposure to illumination for fluorescence, synaptic transmission was unaffected compared with that in controls, and electron microscopy revealed no obvious damage to synaptic structure (S. Karunanithi, L. Marin, and H. L. Atwood, unpublished observations). Scale bar, 10 μm.

Fig. 3.

The success of synaptic transmission at individual boutons is maintained at the higher test temperatures in preparations derived from heat-shocked larvae. The results were obtained from 29 control and 30 heat-shocked neuromuscular junctions. C, Control (open circles);HS_1/2, heat shock followed by a 1/2 hr recovery (filled circles).

Fig. 4.

Postsynaptic stabilization of synaptic performance following heat shock at all test temperatures as revealed by the constant amplitude of mEJCs. mEJC parameters were assessed in control (C, open circles) and heat-shocked (HS_1/2, 1/2 hr recovery,filled circles) experiments at the four test temperatures. Significant differences between the two groups at each test temperature assessed using a t test are denoted with an asterisk. a, Exemplarytraces of mEJCs recorded from individual Ib boutons in control (top) and heat-shocked (bottom) preparations at the four test temperatures. Each record represents an average of three individual events selected close to the mean amplitude. The time course of the unitary events is shortened at the higher test temperature, and the change in amplitude is more pronounced in the control recordings. b, mEJC amplitude. c, Normalized mEJC amplitude. Amplitudes at 27, 31, and 35°C were normalized to that at 22°C in each experiment. Note the larger variance in the control samples.d, mEJC rise times (time to peak). e, mEJC decay time constant obtained by fitting a single exponential to the decay phase of the mEJC. No marked differences in temporal parameters were evident between C andHS_1/2 recordings. Error bars represent SEs.

Fig. 5.

Representative traces of nerve-evoked EJCs recorded from individual Ib boutons in control (C) and heat-shocked (HS_1/2, 1/2 hr recovery) preparations at the four test temperatures. In this experiment, there was no response at 35°C for the control preparation.

Fig. 6.

Presynaptic performance is stabilized after heat pretreatment, but this neuroprotection diminishes with increased recovery time when hsp70 levels are decreased. EJC parameters were assessed in control (C, open circles) and heat shock (HS_1/2, 1/2 hr recovery,filled circles; HS₆, 6 hr recovery, filled squares) experiments at the four test temperatures. Significant differences between C andHS_1/2 at each test temperature assessed using a t test are denoted with anasterisk. a, EJC amplitude.b, Normalized EJC amplitude. Amplitudes at 27, 31, and 35°C were normalized to that at 22°C in each experiment.c, The percentage of transmission failures.d, The estimated mean quantal content. Error bars represent SEs.