Zebrafish model for congenital myasthenic syndrome reveals mechanisms causal to developmental recovery.

Mutations in muscle ACh receptors cause slow-channel syndrome (SCS) and Escobar syndrome, two forms of congenital myasthenia. SCS is a dominant disorder with mutations reported for all receptor subunits except γ. Escobar syndrome is distinct, with mutations located exclusively in γ, and characterized by developmental improvement of muscle function. The zebrafish mutant line, twister, models SCS in terms of a dominant mutation in the α subunit (α(twi)) but shows the behavioral improvement associated with Escobar syndrome. Here, we present a unique electrophysiological study into developmental improvement for a myasthenic syndrome. The embryonic α(twi)ßδγ receptor isoform produces slowly decaying synaptic currents typical of SCS that transit to a much faster decay upon the appearance of adult ε, despite the α(twi) mutation. Thus, the continued expression of α(twi) into adulthood is tolerated because of the ε expression and associated recovery, raising the likelihood of unappreciated myasthenic cases that benefit from the γ-ε switch.

Acetylcholine receptor gating in a zebrafish model for slow-channel syndrome.

Slow-channel syndrome (SCS) is an autosomal-dominant disease resulting from mutations in muscle acetylcholine (ACh) receptor subunits. The associated fatigue and muscle degeneration are proposed to result from prolonged synaptic responses that overload intracellular calcium. Single-channel studies on reconstituted receptors bearing human mutations indicate that the prolonged responses result from an increase in receptor open duration and, in some cases, increased sensitivity to ACh. We show that both of these aberrant receptor properties are recapitulated in heterozygotic zebrafish bearing an L258P mutation in the α subunit, thus affording the unique opportunity to compare the single-channel properties of mutant receptors to the synaptic currents in vivo. Whole-cell recordings revealed synaptic currents that decayed along a multiexponential time course, reflecting receptors containing mixtures of wild-type and mutant α subunits. Treatment with quinidine, an open-channel blocker used to treat the human disorder, restored fast synaptic current kinetics and the ability to swim. Quinidine block also revealed that mutant receptors generate a large steady-state current in the absence of ACh. The spontaneous openings reflected a destabilization of the closed state, leading to an apparent increase in the sensitivity of these receptors to ACh. The effective block by quinidine on synaptic currents as well as nonliganded openings points to dual sources for the calcium-dependent myopathy in certain forms of SCS.

Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.

Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.

Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging.

AIM: Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. RESULTS: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. INNOVATION: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. CONCLUSION: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.

Quantitative two-photon imaging of fluorescent biosensors.

Fluorescent biosensors are now routinely imaged using two-photon microscopy in intact tissue, for instance, in brain slices and brains in living animals. But most studies measure temporal variation-for example, calcium transients in response to neuronal activity-rather than calibrated levels of biosensor occupancy (and thus levels of the sensed analyte). True quantitative measurements are challenging, since it is difficult or impossible to calibrate a sensor's dose-response in situ, and difficult to compare the optical signals from tissue to those during in vitro calibration. Ratiometric measurements (at two wavelengths) are complicated by variations in laser power and by wavelength-dependent attenuation in tissue. For some biosensors, fluorescence lifetime imaging microscopy (FLIM) provides a valuable alternative that gives well-calibrated measurements of analyte levels.

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses.

Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned α(2)ßδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αßδ subunits (presumed to be α(2)ßδ(2) receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of γ/ε-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed α(2)ßδ(2) receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for α(2)ßδ(2) receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down ε subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the α(2)ßδ(2) receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.

Synaptic homeostasis in a zebrafish glial glycine transporter mutant.

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition.