Heterogeneous responses to embryonic critical period perturbations within the Drosophila larval locomotor circuit
- Department of Zoology, University of Cambridge, United Kingdom
- Division of Neuroscience, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, United Kingdom
- Department of Biology, Texas A&M University, United States
Figures
Figure 1
Transient heat stress during the critical period leads to an unstable network.
(A) Acute increase of ambient 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. Left: micrograph of an isolated ventral nerve cord with segmentally repeated aCC motoneurons expressing GCaMP8f. Centre: traces of GCaMP fluorescence peaks measured at different temperatures. Right: quantification of fluorescence peak frequency changes over time (control/sham - bottom trace, n=10 nerve cords) and in response to temperature increase (top trace, n=10 nerve cords). (B) Heat stress, when experienced during the embryonic critical period (CP) of locomotor network development, leads to reduced network stability. Left: Schematic representation of embryonic and larval development and the paradigm of temperature manipulation during embryogenesis. Embryos were kept at the control temperature of 25 °C, either continuously (control) or moved to 32 °C for 2 hr, either before (13–15 hr, 15–17 hr after egg laying, AEL), during (17–19 hr AEL) or after (19–21 hr AEL) the locomotor network CP, then returned to 25 °C until the late larval stage, when subjected to seizure induction by electroshock (n=30 larvae per time point). Right: quantification of seizure recovery times. Data are shown with mean± SEM, ANOVA, ****p<0.00001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 1—source data 1.
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Figure 1—source data 1
Contains numerical data represented graphically in Figure 1.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig1-data1-v1.xlsx
Figure 2
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 6 hr period were incubated at 32 °C for 24 hr to guarantee heat stress exposure during the locomotor network critical period (CP). (B) This results in a change of subsequent neuromuscular junction (NMJ) development, which manifests as a significant overgrowth of aCC presynaptic terminals (green, visualised with anti-HRP-AlexaFluor488). The increase in bouton number and NMJ area is paralleled by concomitant increases in active zones (magenta), resulting in a normal active zone density. (C) Postsynaptically, embryonic heat stress causes a significant decrease of glutamate receptors containing the larger conductance GluRIIA subunit, while those with the GluRIIB subunit remain unaffected. Thus, the postsynaptic GluRII receptor composition is significantly changed following a transient embryonic heat stress, as reflected in the ratio of receptors containing GluRIIA vs GluRIIB. Data points represent measurements from one NMJ per specimen and data are shown with mean ± SEM, unpaired t-test, ***p<0.0001, ****p<0.00001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 2—source data 1.
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Figure 2—source data 1
Contains numerical data represented graphically in Figure 2.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig2-data1-v1.xlsx
Figure 3
Heat stress perturbations during distinct periods of development differentially affect postsynaptic GluRII receptor composition vs neuromuscular junction (NMJ) size.
(A) Schematic of temperature manipulations during embryogenesis. Embryos collected over 1 hr were incubated at 32 °C for different phases of embryogenesis. (B) Resultant changes to the development of the aCC NMJ on muscle dorsal acute muscle 1 (DA1) were quantified at the late wandering third instar larval stage. Reduced expression of the GluRIIA subunit occurred following heat stress experienced during 13–16 hr after egg laying (AEL), comparable to GluRIIA changes caused by heat stress during all of embryogenesis, but not in response to heat stress experienced before or after this 3 hr window. GluRIIB levels are not affected by heat stress during any period or all of embryogenesis. NMJ size, in contrast, is affected by heat stress experienced during earlier phases of muscle development, from 9 to 13 hr AEL. Data are shown with mean ± SEM, ANOVA, *p<0.01, **p<0.001, ***p<0.0001, ****p<0.00001, ‘ns’ indicates statistical non-significance. Sample sizes: for GluRIIA and GluRIIB measurements, each data point represents one NMJ from a unique specimen; for NMJ size, up to four muscle DA1 NMJs in abdominal segments three to five were sampled per larva. For numerical data shown, see Figure 3—source data 1.
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Figure 3—source data 1
Contains numerical data represented graphically in Figure 3.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig3-data1-v1.xlsx
Figure 4
Physiological properties of the late larval neuromuscular junction (NMJ) following an embryonic 32 °C perturbation.
(A) Representative traces of control two-electrode voltage clamp recordings, one per specimen, across a gradient of external calcium concentrations. (B) Postsynaptic current amplitude is not altered between control and embryonic heat stress-manipulated animals at any given external calcium concentration (n=5). (C) Plotting postsynaptic current amplitude against its variance to calculate quantal size (q) and the number of release sites being used (N), also did not show a significant difference between control and embryonic heat stress-manipulated animals (n=5). (D) To measure readily releasable pool size, a 60 Hz stimulus train was applied for 1 s 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 (n=9) as compared to controls (n=11). (E) Cumulative amplitude was plotted over time and a linear regression was back extrapolated through the last 20 pulses (same data as for E). (F) The y intercept was divided by the Mini size of the same trace to calculate the readily releasable pool (RRP) size. (G) NMJs in heat stress-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 manipulated animals. Data in F & G are shown with mean ± SEM, ANOVA, *p<0.01, **p<0.001, ***p<0.0001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 4—source data 1.
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Figure 4—source data 1
Contains numerical data represented graphically in Figure 4.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig4-data1-v1.xlsx
Figure 5
Larval motoneurons display reduced excitability and increased synaptic input after an embryonic 32 °C heat stress experience.
(A) Embryos were subjected to 32 °C heat stress, then reared to the late larval stage under control conditions of 25 °C. Whole-cell patch clamp recordings show action potentials generated by aCC motoneurons isolated from excitatory synaptic input by mecamylamine addition in response to current injection. Inset shows linear regression centred around 32–72 pA current injections, showing significant reduction in excitability in heat stress manipulated specimens relative to controls. (B) Whole-cell patch clamp recordings from the aCC motoneuron in larvae: (Bi) reveal an increase in the duration of spontaneous rhythmic currents (SRCs) in larvae that had previously experienced 32 °C heat stress during embryonic development. Current density and frequency of SRCs remain comparable to controls. (Bii) Targeted optogenetic activation (using Chronos, for 1 s) of one premotor excitatory interneuron, A27h, showed A27h synaptic input has increased as a result of a previous embryonic 32 °C heat stress experience. Each data point represents information recorded from a single neuron per specimen. Data are shown with mean ± SEM, unpaired t-test, *p<0.01, ****p<0.00001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 5—source data 1.
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Figure 5—source data 1
Contains numerical data represented graphically in Figure 5.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig5-data1-v1.xlsx
Figure 6
Spontaneous larval motoneuron and muscle firing properties are unchanged following embryonic heat stress.
(A) Monitoring spontaneous motoneuron firing by cell-attached patch clamp recordings from the aCC motoneuron in a late third instar larval nerve cord found no significant differences between embryonically manipulated specimens and controls, regarding burst rate per second, burst duration or number of spikes per burst (n=10). (B) Measuring muscle output generated by motoneuron spontaneous firing with current clamp recordings from the dorsal acute muscle 1 (DA1) muscle in late third instar larvae showed that larvae are able to produce normal motoneuron firing output to the muscle, irrespective of embryonic experience (n=8 for controls, n=6 for experimentals with 32 °C embryonic experience). Each data point represents information recorded from a single neuron per specimen in (A), or neuromuscular junction (NMJ) per larva in (B). Data are shown with mean ± SEM, unpaired t-test, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 6—source data 1.
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Figure 6—source data 1
Contains numerical data represented graphically in Figure 6.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig6-data1-v1.xlsx
Figure 7
Embryonic heat stress manipulation causes reduced speed of both 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 embryonic development. Activity wave propagation across abdominal segments is slowed down, i.e., intersegment duration has increased, notably between anterior segments, A3 to A1. Each data point represents a unique specimen. (B) In intact animals, locomotor network output is similarly altered: embryonic heat stress (32°C) leads to a significant reduction in larval crawling speed, as compared to controls (25°C). Crawling speed was measured in >20 larvae per condition, with up to three data points obtained per larva, each from a distinct 5 min recording. Data are shown with mean ± SEM, unpaired t-test, **p<0.001, ****p<0.00001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 7—source data 1.
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Figure 7—source data 1
Contains numerical data represented graphically in Figure 7.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig7-data1-v1.xlsx
Figure 8
Larval crawling speed is determined by the critical period (CP) of central locomotor network.
(A) Embryos were subjected to heat stress (32 °C) during different windows of development. Crawling speed of resultant third instar larvae was affected only when the CP for the central locomotor network (17–19 hr after egg laying [AEL]) was manipulated. (B) Embryonic experience of heat stress leads to larvae that retain their ability to increase crawling speed in response to acute relative increases in ambient temperature, to a higher absolute temperature (35 °C) than controls (29 °C) >20 larvae were sampled per condition, each sampled up to three times, once per unique 5 min crawling interval. Data are shown with mean ± SEM, ANOVA, *p<0.01, **p<0.001, ****p<0.00001, ‘ns’ indicates statistical non-significance. For numerical data shown, see Figure 8—source data 1.
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Figure 8—source data 1
Contains numerical data represented graphically in Figure 8.
- https://cdn.elifesciences.org/articles/108656/elife-108656-fig8-data1-v1.xlsx
Figure 9
Model of distinct critical periods for body wall muscles and the central locomotor circuitry.
Embryonic heat stress (32 °C) leads to adjustments of postsynaptic receptor fields, when applied during an earlier developmental window of 13–16 hr after egg laying. Subsequently, from 17 to 19 hr after egg laying the central locomotor circuitry is sensitive to perturbations, leading to an increase in synaptic drive of the premotor network, potentially including central pattern generating circuits (CPGs). The working model posits that in response, motoneurons decrease their excitable properties, thus maintaining normal locomotor output to the body wall muscles. Nevertheless, changes in the premotor network lead to a reduction in activity wave progression and thus reduced larval crawling speed, also manifesting a reduction in overall network stability. Speculatively, preferential maintenance of GluRIIA in distal boutons might have explained maintenance of normal synaptic transmission at low-frequency stimulation regimes. However, we found no evidence for this. On the contrary, in embryonic heat stress-manipulated specimens, GluRIIA levels were comparably reduced in both proximal and distal boutons (data not shown). An alternative explanation for maintained synaptic transmission in the face of increased active zone number would be a reduction in release probability. Although we did not conduct functional imaging to investigate this directly, an indirect indication for this is the increased facilitation in animals that had experienced embryonic heat stress, which indicates a reduction in presynaptic release probability across the whole neuromuscular junction (NMJ) (Figure 4D).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | Oregon-R | Bloomington Drosophila Stock Center | RRID:BDSC_2376 FLYB: FBst0002376 | wild-type control |
| Strain, strain background (D. melanogaster) | Canton S | Bloomington Drosophila Stock Center | RRID:BDSC_64349 FLYB: FBst0064349 | wild-type control |
| Genetic reagent (D. melanogaster) | GMR94G06-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_40701 FLYB: FBti0139793 | FlyBase symbol: w1118;; GMR94G06-GAL4 [attP2] |
| Genetic reagent (D. melanogaster) | A27h-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_49939 FLYB: FBti0135233 | FlyBase symbol: w1118;; P{y[+t7.7] w[+mC]=GMR36 G02-GAL4}attP2 |
| Genetic reagent (D. melanogaster) | A27h-split-Gal4 | Created by A.A. Zarin, this study. Components available in Bloomington Drosophila Stock Center | RRID:BDSC_70953 FLYB: FBti0188221 RRID:BDSC_68768 FLYB: FBti0191234 | FlyBase symbol: w1118; 34F03-AD [attP40]; 36G02-DBD [attP2] |
| Genetic reagent (D. melanogaster) | 20XUAS-IVS-jGCaMP8f | Bloomington Drosophila Stock Center | RRID:BDSC_92587 FLYB: FBti0215881 | FlyBase symbol: w1118;; 20XUAS-IVS-jGCaMP8f [VK00005] |
| Genetic reagent (D. melanogaster) | 20XUAS-Chronos-mVenus | Bloomington Drosophila Stock Center | RRID:BDSC_77115 FLYB: FBti0195682 | FlyBase symbol: w1118; 20XUAS-Chronos-mVenus [attP40] |
| Antibody | anti-Bruchpilot (NC82) (Mouse monoclonal) | DSHB | Cat# nc82, RRID:AB_2314866 | IF(1:100) |
| Antibody | anti-DGluRIIA 8B4D2 (MH2B) (Mouse monoclonal) | DSHB | Cat# 8B4D2 (MH2B), RRID:AB_528269 | IF(1:100) |
| Antibody | anti-DGluRIIB (Rabbit polyclonal) | Mihaela Serpe (Sulkowski et al., 2016) | IF(1:1000) | |
| Antibody | anti-mouse- STAR Red (Goat polyclonal) | Abberior | Cat#: STRED-1001–500 UG; RRID:AB_3068620 | IF(1:1000) |
| Antibody | anti-rabbit- ATTO594 (Goat polyclonal) | Hypermol | Cat#: 2306; | IF(1:1000) |
| Antibody | Alexa Fluor488 Goat Anti-Horseradish Peroxidase (Goat polyclonal) | Jackson ImmunoResearch | Cat#: 123-545-021; RRID:AB_2338965 | IF(1:1000) |
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Heterogeneous responses to embryonic critical period perturbations within the Drosophila larval locomotor circuit
eLife 14:RP108656.
https://doi.org/10.7554/eLife.108656.3
