Transient heat stress during the critical period leads to unstable networks.

A Schematic representation of embryonic and larval development and temperature manipulation during embryogenesis. Embryos were kept at 32°C for 2 h either before (13-15 h, 15-17 h AEL), during (17-19 h AEL) or after (19-21 h AEL) the locomotor network CP. B Acute increase of temperature leads to increased network activity, as measured with the fluorescent calcium indicator GCaMP8f expressed in segmentally repeated aCC motoneurons in isolated central nervous systems; while constant 25°C leads to reduced activity over time.

Morphological changes at the neuromuscular junction after critical period perturbation.

A) Schematic of temperature manipulation during embryogenesis and location of dorsal acute muscle 1 (DA1) within a fillet dissected larva. Embryos collected over a 6h period were incubated at 32°C for 24h to guarantee heat stress exposure during the embryonic CP, 17-19 hours after egg laying. B) This results in a change of subsequent NMJ development with significant overgrowth of aCC presynaptic terminals (green) and an increase in bouton number and NMJ area. A concomitant increase in active zone number (magenta) results in a normal active zone density. C) Postsynaptically, CP heat stress result in a decrease of GluRIIA (Ci) while GluRIIB levels remain unaffected (Cii).

Physiological NMJ properties after 32°C perturbation.

A) Representative traces of control two electrode voltage clamp recordings across a gradient of external calcium concentrations. B) Postsynaptic current amplitude is not altered between control and CP-manipulated animals at any given external calcium concentration. C) Plotting postsynaptic current amplitude against its variety to calculate quantal size (q) and the number of release sites being used (N), also did not show a significant difference between control and CP-manipulated animals. To measure readily releasable pool size, a 60Hz stimulus train was applied for 1s (D) where we could observe a slight increase in facilitation over the first 10 pulses in specimens that in their embryonic stage had experienced heat stress of 32°C. Cumulative amplitude was plotted over time and a linear regression was back extrapolated through the last 20 pulses (E). The y intercept was divided by the Mini size of the same trace to calculate the readily releasable pool size. G-Giii) NMJs in CP-manipulated specimens have normal EPSC and mEPSC amplitudes and quantal content. Acute application of the GluRIIA blocker PhTx reliably induced presynaptic homeostatic potentiation in controls and 32°C CP-manipulated animals.

Larval motoneurons display reduced excitability and increased synaptic input after an embryonic 32°C CP-manipulation.

A aCC motoneurons at the late third instar larval stage, isolated from excitatory synaptic input. Motoneurons in specimens, which during the embryonic CP had transiently experienced 32°C heat stress, show significant reduction in excitability relative to controls. B Left: whole cell patch clamp recordings from the aCC motoneuron reveal an increase in spontaneous rhythmic current duration several days after embryos had experienced transient 32°C heat stress during the CP. Right: targeted optogenetic activation (using Chronos) of a premotor excitatory interneuron (A27h) showed that A27h input is increased in late larvae that as embryos had experienced 32°C heat stress during the CP.

Spontaneous larval motoneuron and muscle firing properties and are unchanged after embryonic temperature challenge.

A Monitoring spontaneous firing by loose cell patch clamp recordings from the aCC motoneuron in a late third instar larval nerve cord did not show a significant difference between CP-manipulated specimens and controls. B Measuring muscle output of motoneuron spontaneous firing by current clamp recordings from the DA1 muscle in late third instar larvae showed that animals are able to produce normal motoneuron firing output to the muscle, irrespective of embryonic experience.

CP manipulation causes reduced speed of activity wave propagation and larval crawling.

A Functional imaging of A27h premotor interneurons using GCaMP8f in isolated CNSs from late larvae that previously had experienced transient 32°C heat stress during their embryonic CP. Propagation of the activity wave is slowed down, due to an increase in intersegment duration, notably between anterior segments, e.g. A3 and A1. B In intact animals, network activity is similarly altered: CP heat stress (32°C) leads to a significant reduction in larval crawling speed, as compared to controls (25°C).