bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.08.30.610471

Novel iGluSnFR3 variants with improved kinetics and dynamic range for tracking high frequency firing at glutamatergic synapses

Tran, O.·Bertelli, S.·Hughes, H.·Lozo, L.·Plested, A.·Torok, K.
View original source ↗Export metadata ↓

Abstract

The genetically-encoded fluorescent glutamate sensor, iGluSnFR3, characterised by a high dynamic range and rapid on-kinetics, is an attractive sensor for glutamate imaging in the central nervous system. However, kinetic variants with ultrafast or slow off-kinetics are needed to broaden the spectrum of applications from monitoring rapid synaptic glutamate transients to mesoscale imaging of brain activity. Here we report binding-site variant S72T (iGlu3Fast) optimised for reporting fast glutamate release at individual sites with a fluorescence dynamic range of 57 and a decay t1/2 of 2 ms in solution, [~]5-fold faster than iGluSnFR3. In contrast, variants D25A and D25R (termed iGlu3Slow1 and iGlu3Slow2, respectively) presented with slow off-kinetics (decay t1/2 43 ms and 30 ms, respectively, at 20{degrees}C), while retaining high dynamic ranges (48 and 65, respectively). These values were reduced when expressed in HEK293T cells, glutamate dissociation from iGlu3Fast slowed to a decay t1/2 of 6.9 ms, while decay t1/2-s of 115 and 234 ms were measured for iGlu3Slow1 and iGlu3Slow2, respectively. The slow decay rates in cells were in accordance with reduced Kd-s in the range of 200 M for iGlu3Fast and 6.4 and 8.4 M for iGlu3Slow1 and iGlu3Slow2, respectively. 2-photon imaging in organotypic hippocampal slices reported spontaneous activity with high sensitivity by both iGlu3Fast and iGlu3Slow2. Strikingly, different kinds of glutamate transient were detected by the fast- and slow-decay sensors indicating that the rapid kinetics and increased fluorescence dynamic range make iGlu3Fast an excellent candidate for imaging high-frequency glutamate release at synapses while iGlu3Slow2 has the potential for mesoscale imaging of brain activity to record global events with high sensitivity.

Explore related subjects

neurosciencePreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Tran, O., Bertelli, S., Hughes, H., Lozo, L., Plested, A., Torok, K.. 2024-08-30. Novel iGluSnFR3 variants with improved kinetics and dynamic range for tracking high frequency firing at glutamatergic synapses. https://doi.org/10.1101/2024.08.30.610471

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A systems-level model of sleep-dependent memory-consolidation failure in neurodegeneration: the spindle-slow-oscillation decoupling cascade dissociates amyloid and tau

During non-rapid-eye-movement (NREM) sleep, the temporal coupling of cortical slow oscillations (SOs), thalamic spindles, and hippocampal sharp wave ripples drives the consolidation of declarative memories. This coupling degrades in ageing and Alzheimers disease (AD), and although A{beta} and tau leave dissociable signatures in human sleep, the mechanisms by which progressive pathology dismantles the consolidation machinery are difficult to isolate experimentally, and have not to our knowledge been reproduced in a model that can be perturbed directly. We built a systems-level model in which cortical SOs and thalamic spindles are generated by reduced oscillators, hippocampal ripples replay encoded spike sequences, and the measured per-event SO-spindle timing alignment causally gates spike-timing dependent plasticity on cortical sequence synapses. A post-sleep cued-recall test reads out consolidation. Five neurodegeneration parameters (amyloid, tau, synaptic density, GABAergic inhibition, cholinergic tone) map to dis tinct mechanisms grounded in the human and animal literature. The model reproduces graded healthy consolidation and a progressive collapse in which coupling, slow-wave power, spindle power and recall fall monotonically and the overnight memory effect flips from consolidation to net forgetting, with weak memories failing first. Scrambling SO-spindle timing while holding oscillation power fixed abolishes consolidation, establishing that coupling timing, rather than oscillation power, is what the plasticity gate depends on within the model. A{beta} and tau impair memory through orthogonal signatures (A{beta} collapses slow-wave power while sparing replay order, tau the reverse) and this orthogonality holds across the entire A{beta} x tau plane and survives simultaneous {+/-}50% resampling of every mapping coefficient (40/40 samples), so it is not an artefact of a single calibration point. The model yields a falsifiable clinical prediction: closed-loop slow-oscillation enhancement rescues memory only when the deficit is amplitude/coupling-dominated, not when it is replay(tau)-dominated, despite normalising slow-wave power in both cases. Because the therapy arms dissociate coupling from memory benefit, the model also cautions against adopting SO-spindle coupling as a standalone surrogate endpoint.

neuroscience↗
bioRxiv2026

Toxicity of MAPT 4R RNA Contributes to Motor Neuron Degeneration in ALS

MAPT (Tau) dysregulation is implicated in several neurodegenerative diseases, but its contribution to amyotrophic lateral sclerosis (ALS) is poorly understood. Here we show that mRNA isoforms encoding 4-repeat (4R) Tau are upregulated and cytoplasmically enriched in iPSC-derived motor neurons (MNs) from VCP-mutant and sporadic ALS, without a corresponding change in Tau protein. Using splice-switching antisense oligonucleotides and isoform-specific siRNAs, we find that enhanced 4R expression reduces MN viability, whereas its selective knockdown improves survival, with kinetics more consistent with an RNA-intrinsic effect than altered protein synthesis. Exon 10-containing MAPT RNA shows increased predicted secondary structure, self-association and altered Tau biocondensation in vitro. In post-mortem ALS cervical spinal cord, increased relative exon 10 usage is associated with a higher-risk clinical phenotype and shorter disease duration These findings identify an isoform-specific contribution of MAPT to MN vulnerability in ALS and nominate 4R MAPT RNA as a therapeutic target.

neuroscience↗
bioRxiv2026

State dependant modulation of optic flow-processing lobula plate cells in butterflies

Increasing experimental evidence suggests that biological systems cancel predictable components of sensory signals while maintaining sensitivity to externally induced state changes. This strategy provides task-specific sensor responses for posture, locomotion, and gaze control. A prime example is found in interneurons that respond to visual image shifts resulting from the relative motion between an animal's eyes and its visual surroundings. Such optic flow-processing interneurons, found across phyla and are particularly well characterized in Dipteran and other flying insects. We studied optic flow-processing interneurons in the Monarch butterfly whose large and highly contrasted wings sweep through the visual field with every wing-beat cycle, potentially obscuring interneuron output signals. Our results show baseline spiking activity increases when animals flap their wings, and individual spikes are phase-locked to the wing-beat cycle, even in the dark, when no visual motion input is available. A qualitative estimate of the interneurons' response to directional wing motion through its receptive field is not sufficient to explain the recorded activity patterns. Our results suggest that an additional internal signal suppresses responses to wing-induced visual motion to support effective vision-based stabilization reflexes. These findings support the principle that self-generated signals are suppressed while sensitivity to external modulation is preserved.

neuroscience↗