Biology subjects

Torok, K.

Publications and source records attributed to Torok, K..

4 recordsLinked to original sources

bioRxiv2024

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

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.

neuroscience↗

bioRxiv2019

Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding: affinity variants of iGluSnFR termed iGluh, iGlum and iGlul covering a range of Kd-s (5.8 M, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with a Kd of 9.7 M are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluu and iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluh and Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.\n\nSTATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.

biophysics↗

bioRxiv2018

Glutamate uptake and release at single adult corticostriatal synapses of normal and Huntington mice

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGluu. We report findings from individual corticostriatal synapses in acute slices prepared from mice aged >1 year. Contrasting patterns of short-term plasticity and a size criterion identified 2 classes of terminals, presumably corresponding to the previously defined IT and PT synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGluu single synapse study in Q175 mice, a model of Huntingtons disease (HD). It was found that in HD the time constant of perisynaptic [Glu] decay (TauD, as indicator of uptake) and the peak iGluu amplitude (as indicator of release) were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nM) mimicked the TauD changes in homozygotes (HOM). Considering the largest TauD values encountered in WT, about 40% of PT terminals tested in Q175 heterozygotes (HET) can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGluu. Finally, EAAT2 immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention. SIGNIFICANCE STATEMENTAlterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor [Glu] at individual corticostriatal synapses we can now quantify the time constant of perisynaptic [Glu] decay (as indicator of uptake) and the maximal [Glu] elevation next to the active zone (as indicator of Glu release). The results provide a positive answer to the hitherto unresolved question whether neurodegeneration (e.g. Huntingtons disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

neuroscience↗

bioRxiv2017

Ultrafast glutamate sensors resolve synaptic short-term plasticity

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to [~]10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 M and 600 M, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.\n\nSignificance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

neuroscience↗