Differences in Ca2+ regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae

doi:10.1016/j.neuroscience.2009.01.074

Neuroscience

Volume 159, Issue 4, 10 April 2009, Pages 1283-1291

https://doi.org/10.1016/j.neuroscience.2009.01.074 Get rights and content

Abstract

We determined whether two classes of Drosophila larval motor terminals with known differences in structure and transmitter release also showed differences in Ca²⁺ regulation. Larval motor neurons can be separated into those producing large synaptic boutons (Ib) and those with small boutons (Is). Ib terminals release less transmitter during single action potentials (APs) than Is terminals, but show greater facilitation during high-frequency stimulation. We measured Ca²⁺ transients produced by single APs and AP trains after loading the terminals with the dextran-conjugated Ca²⁺ indicator Oregon Green 488 BAPTA-1 (OGB-1). The two pairs of Is and Ib terminals innervating muscle fiber 4 and fibers 6 and 7 were examined. The OGB-1 concentrations were measured in order to compare measurements from terminals with similar OGB-1 loading. For single APs, the change in OGB-1 fluorescence (ΔF/F) in Is boutons was significantly larger than in Ib boutons due to greater Ca²⁺ influx per bouton volume. The Is boutons had greater surface area and active zone number per bouton volume than Ib boutons; this could account for the differences in Ca²⁺ influx and argues for similar Ca²⁺ influx at Is and Ib active zones. As previously reported for the Ib boutons, the distal Is boutons had larger single-AP Ca²⁺ transients than proximal ones on muscle fibers 6 and 7, but not on fiber 4. This difference was not due to proximal–distal differences in surface area or active zones per bouton volume and may be due to greater Ca²⁺ influx at distal active zones. During AP trains, the Is Ca²⁺ transients were larger in amplitude and had longer decay time constants than Ib ones. This can be explained by a slower rate of Ca²⁺ extrusion from the Is boutons apparently due to lower plasma membrane Ca²⁺ ATPase activity at Is boutons compared to Ib boutons.

Section snippets

Loading the terminals with OGB-1

All experiments were performed on the terminals innervating MFs 6 and 7 (MF 6/7) and those innervating MF 4 in segment 3 of Drosophila (Canton S) wandering 3rd-instar, female larvae. To measure changes in [Ca²⁺]_i, we used the single-wavelength Ca²⁺ indicator, Oregon Green 488 BAPTA-1 (OGB-1) coupled to 10,000 MW dextrans; its dissociation constant (K_d) was 1180 nM as reported by Molecular Probes (Invitrogen, Carlsbad, CA, USA). The technique for loading the indicator was developed by Macleod et

Is and Ib terminals show differences in Ca²⁺ transients

To measure Ca²⁺ transients, the terminals were loaded with a dextran conjugate of OGB-1. The OGB-1 reported changes in cytosolic [Ca²⁺] since the dextran prevented compartmentalization of the OGB-1. We measured Ca²⁺ transients produced by single APs and 10 Hz trains of APs in the two pairs of Is and Ib terminals innervating MF 4 and 6/7. These four terminals are all generated by different axons (Lnenicka and Keshishian 2000, Hoang and Chiba 2001). We have found that MF 4 occasionally has two Ib

Is–Ib differences in Ca²⁺ transients

We measured ΔF/F by capturing images of the terminals during nerve stimulation and found that the Ca²⁺ transients were larger for Is than Ib boutons. The ΔF/F_AP peak and ΔF/F_train plateau were greater at Is boutons than at Ib boutons for both MFs 4 and 6/7. Measurements of ΔF/F also showed differences in the τ_decay for Is and Ib boutons. The ΔF/F_AP τ_decay was longer for Is boutons than for Ib boutons on MF 4, but not on MF 6/7. For AP trains, the τ_decay was longer for Is boutons than for Ib

Acknowledgments

Supported by NSF grant IOB 0543835 (G.A.L.).

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Finally, the Ca2+ signals evoked by 10 and 20 Hz stimulus trains were no different in neto-αnull (10 Hz, data not shown; Ib, p = 0.63; Is, p = 0.93) (20 Hz; Ib, p = 0.37; Is, p = 0.40) (Figure 6E). Although the data shown here are sufficiently sensitive to reveal the differences in Ca2+ entry known to exist between type Ib and type Is terminals (He et al., 2009; Lu et al., 2016), they reveal no deficit in Ca2+ entry in neto-αnull terminals. Thus, neto-αnull neurotransmission deficits are most likely the result of deficits in the release apparatus downstream of Ca2+ entry.
Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.
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In practice, this approach depends on a favorable nerve terminal architecture. It has so far been possible to detect activity-dependent Ca2+ transients in motor nerve terminals of Drosophila (He et al., 2009a,b; Macleod et al., 2002), crayfish (Delaney et al., 1989; He et al., 2009b; Lnenicka et al., 2006; Millar et al., 2005; Msghina et al., 1999; Tank et al., 1995), frogs (DiGregorio et al., 1999; Luo et al., 2011; Wachman et al., 2004) and lizards (David et al., 1997). ( The geometric complexity of nematode and mammalian NMJs has so far prevented similar studies in these animals).
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Fragile X syndrome (FXS) is a broad-spectrum neurological disorder characterized by hypersensitivity to sensory stimuli, hyperactivity and severe cognitive impairment. FXS is caused by loss of the fragile X mental retardation 1 (FMR1) gene, whose FMRP product regulates mRNA translation downstream of synaptic activity to modulate changes in synaptic architecture, function and plasticity. Null Drosophila FMR1 (dfmr1) mutants exhibit reduced learning and loss of protein synthesis-dependent memory consolidation, which is dependent on the brain mushroom body (MB) learning and memory center. We targeted a transgenic GFP-based calcium reporter to the MB in order to analyze calcium dynamics downstream of neuronal activation. In the dfmr1 null MB, there was significant augmentation of the calcium transients induced by membrane depolarization, as well as elevated release of calcium from intracellular organelle stores. The severity of these calcium signaling defects increased with developmental age, although early stages were characterized by highly variable, low fidelity calcium regulation. At the single neuron level, both calcium transient and calcium store release defects were exhibited by dfmr1 null MB neurons in primary culture. Null dfmr1 mutants exhibit reduced brain mRNA expression of calcium-binding proteins, including calcium buffers calmodulin and calbindin, predicting that the inability to appropriately sequester cytosolic calcium may be the common mechanistic defect causing calcium accumulation following both influx and store release. Changes in the magnitude and fidelity of calcium signals in the absence of dFMRP likely contribute to defects in neuronal structure/function, leading to the hallmark learning and memory dysfunction of FXS.
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Prolonged exposure to inorganic lead (Pb²⁺) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). To explore this possibility, we studied the effect of Pb²⁺ exposure on [Ca²⁺]_i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb²⁺ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca²⁺]_i and the changes in [Ca²⁺]_i produced by impulse activity, the motor terminals were loaded with a Ca²⁺ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb²⁺ did not significantly change the resting [Ca²⁺]_i nor the Ca²⁺ transient produced in synaptic boutons by single action potentials (APs); however, the Ca²⁺ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb²⁺-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb²⁺, the increase in [Ca²⁺]_i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca²⁺]_i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca²⁺ ATPase (PMCA), which extrudes Ca²⁺ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb²⁺-dependent reduction in PMCA activity. We also observed a Pb²⁺-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb²⁺-reared larvae than in controls. These results showed that Pb²⁺ exposure produced changes in the regulation of [Ca²⁺]_i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca²⁺]_i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.
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Cellular NeuroscienceDifferences in Ca2+ regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae

Abstract

Section snippets

Loading the terminals with OGB-1

Is and Ib terminals show differences in Ca2+ transients

Is–Ib differences in Ca2+ transients

Acknowledgments

Int Rev Neurobiol

Neuron

Biophys J

Dev Biol

Neuropharmacology

Neuron

Int Rev Neurobiol

Neuron

Neuron

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

J Neurobiol

Strength of synaptic transmission at neuromuscular junctions of crustaceans and insects in relation to calcium entry

Invert Neurosci

Standard mathematical tables and formulae

Activity-dependent plasticity of calcium clearance from crayfish motor axons

J Neurophysiol

Heterogeneity in synaptic transmission along a Drosophila larval motor axon

Nat Neurosci

Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus

J Neurosci

Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos

Proc Natl Acad Sci U S A

Imaging of calcium in Drosophila larval motor nerve terminals

J Neurophysiol

The drosophila neuromuscular junction: a model system for studying synaptic development and function

Annu Rev Neurosci

Cellular Neuroscience
Differences in Ca²⁺ regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae

Is and Ib terminals show differences in Ca²⁺ transients

Is–Ib differences in Ca²⁺ transients