optical reporter of synaptically released glutamate (iglusnfr)  (Nikon)

Journal: bioRxiv

Article Title: Optical analysis of the action range of glutamate in the neuropil

doi: 10.1101/2021.02.05.429974

Figure Lengend Snippet: (A) Cartoon illustrating the recording condition to quantify the spatial spread of synaptically released glutamate. A granule cell (gc) was patch clamped and dye-filled (red) to identify a synaptic bouton (mossy fiber bouton, mfb) surrounded by neuronal iGluSnFr expression (green). To visualize synaptic glutamate release, a 2P line scan was drawn through the bouton (blue line). The boxed region represents a typical frame scan (as illustrated in lower panel) obtained to identify boutons and adjust the line scan. Lower panel, example dual channel two photon frame scan of a dye-filled (TMR 400 µM, red, iGluSnfr, green) mfb used for stimulation and recording of synaptic glutamate release (as shown in I-L). The bath solution contained CNQX (10 µM) to eliminate network activity and 4-AP (100 µM) and DPCPX (1 µM) to elevate release probability and thereby shorten the required recording time (release probability normally below 10%). (B) Dual channel 2P line scan through the bouton shown in A). Top panel shows a failure, bottom panel successful glutamate release, respectively. White lines illustrate the corresponding whole cell current clamp recordings of the stimulated action potentials. The bouton is located in the red channel displayed on the left and does not show changes in fluorescence (tracer dye). In each line scan image, the region between the two dashed grey lines was used to calculate the fluorescence over time traces shown in C). Note the rapidly rising signal only occurring at the position of the bouton and at the time of the action potential. Scale bar: 50 ms, 1 µm. (C) Top panel, 30 example traces of line scan fluorescence over time (as indicated in B)) demonstrate the well-known typical fluctuation of responses (red) and failures (black) known from synaptic vesicle release. Asterisk, time of action potential. Fluorescence normalized to pre-stimulus levels. Bottom panel, peak amplitudes of the fluorescence traces for the 30 sequential stimulations obtained from this bouton. Markers below the dashed horizontal line represent events putatively classified as single vesicle release responses. (D) Line scans of synaptic responses only (excluding release failures) were averaged per bouton to improve signal-to-noise ratio for quantification of the spread of synaptically released glutamate. Grey dashed lines indicate distances from the center of release. (E) Fluorescence over time extracted from D) at the indicated distances. Note that the decay is slowed with distance and that weak signals can still be detected at 2 µm. The peak of these signals was quantified and plotted in F). (F) Synaptically released glutamate activated iGluSnFr at distances of more than 1.5 µm (n=6). In each experiment the peak amplitudes of fluorescent transients were normalized to the largest amplitude measured at the dye-filled bouton.

Article Snippet: Imaging data for the optical reporter of synaptically released glutamate (iGluSnFr) was acquired on a Nikon A1R MP 2-photon scanning microscope (Nikon) equipped with a BVC-700 (Dagan) amplifier and using WinWCP software (Strathclyde) for current clamp recording.

Techniques: Expressing, Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Optical analysis of the action range of glutamate in the neuropil

doi: 10.1101/2021.02.05.429974

Figure Lengend Snippet: (A)Spatial extent of small synaptic iGluSnFr transients. For each recording the smallest events were selected to exclude potential multi-quantal events. Note that the lambda value is in the same range as the one derived from data obtained by averaging small and large transients (cf. ). (B) iGluSnFr fluorescent traces, the selected fraction of traces used for A) is shown in black. Traces are peak-scaled for comparison are not shown at the same vertical scaling. Events for the cell at the right top are shown in . (C) Example 2P line scan across a dye filled bouton showing the action potential-elicited iGluSnFr response (arrowhead), and 2 spontaneous, off-bouton events (black asterisks) used for the analysis shown in D). The white trace represents the simultaneous current clamp recording of the cell stimulated to fire an action potential which released transmitter at the arrow head position (scale bars: 20 mV, 50 ms). Right panel illustrates fluorescent example traces calculated at the positions indicated by the symbols. (D) Spatial extent of spontaneous likely miniature glutamate transients. Events were analyzed that occurred independently of the timing of the action potential induced in the patch-clamped granule cell and all of them must have been released from neighboring synapses because they did not occur at the dye filled bouton. As spontaneous action potential firing of granule cells in slices is very rare and slices are also bathed in CNQX and APV, these events are likely due to miniature, action potential-independent, single vesicle glutamate release. (E) λ sniffer only weakly depends on the magnitude of the signals and tends to be larger if more glutamate is released. From left to right: selected small, spontaneous and evoked events.

Article Snippet: Imaging data for the optical reporter of synaptically released glutamate (iGluSnFr) was acquired on a Nikon A1R MP 2-photon scanning microscope (Nikon) equipped with a BVC-700 (Dagan) amplifier and using WinWCP software (Strathclyde) for current clamp recording.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Optical analysis of the action range of glutamate in the neuropil

doi: 10.1101/2021.02.05.429974

Figure Lengend Snippet: (A)iGluSnFr reports a similar spread of extracellular glutamate following 2P-glutamate uncaging. Cartoon: yellow circle indicates glutamate uncaging site in the dendritic region of CA1 (Str. radiatum) where iGluSnFr reporter proteins are expressed on neuronal membrane (green dots). 2P line scans perpendicular or parallel to axons (blue lines) were used to quantify the spatial spread of the fluorescent signal. Middle panel: Example line scans through the glutamate uncaging site (green asterisk, indicating time and position, average of 3 repeated uncaging spots at 3 s intervals). Note the rapid and substantial spread of the fluorescence. Line scans were normalized on the pre-uncaging fluorescence to account for spatial variability of initial iGluSnFr brightness (owing to varying spatial densities of membrane expression levels). Right panel: example fluorescent traces calculated from the line scan image shown in the middle. Numbers indicate distance from uncaging site; asterisk, time of uncaging. Note the visible and delayed signal at ± 3 µm. Kinetics and amplitude are similar to synaptically evoked iGluSnFr responses as illustrated by the pink trace, average response from the experiment shown in . Scale bar: 100 ms, 100% (B) λ sniff_unc measured from iGluSnfr signals is isotropic (n=10 for each direction, black and grey markers represent scans parallel and perpendicular to axons, respectively) and only slightly exceeds λ sniff_syn obtained following synaptic glutamate release. (C) 2P scan of a dye-filled spine incubated in 20 µM CNQX and 1 µM TTX to isolate NMDA receptors. Uncaging spots (green) were separated by 500 nm and applied at 5 s intervals to account for the substantially slower kinetics of NMDA-R mediated uEPSC. Lower panel: λ NMDA after glutamate uncaging (n=12). (D) Example traces of NMDA receptor-mediated uEPSCs (asterisk, time of uncaging, cell voltage clamped at +40 mV). uEPSCs are still clearly seen at a distance of 2 µm and their kinetics are substantially slower. To reliably quantify peak amplitudes of even the smallest responses uEPSCs were fitted with a two-exponential function (grey line, see methods). Note that even remotely evoked uEPSCs (>1500 nm) evoke clear currents demonstrating pronounced diffusional propagation of released glutamate. (E) Widespread activation of PSD95-GCamp6F following a single uncaging pulse confirms large action range of glutamate at NMDA receptors. Three 2-photon scans (take from the 20 Hz time series quantified in F)) in the dendritic region of CA1 before and after the uncaging pulse (green circle indicates uncaging site, 15 µM glycine to allow NMDA-R activation at resting potential, 20 µM CNQX, 1 µM TTX). Note the appearance of bright spine head-shaped structures following glutamate uncaging which occur even outside a 2 µm range (grey dashed circles). Colored squares indicate example ROIs used to calculate the fluorescence over time traces displayed in F). (F) Average ROI fluorescence over time illustrating the pronounced calcium increases induced in spine heads by activation of NMDA receptors following glutamate uncaging (asterisk, colors of traces correspond to the ROIs shown in E). (G)Estimation of λ NMDA from the spatial distribution of calcium responses (PSD95-GCamp6F) around the uncaging point. The histogram plots the frequency of responding pixels (see methods for threshold details) along the radial distance from the uncaging site (black bars, “responding”, aggregated results over 66 uncaging events). The white bars show the number of pixels in the acquired image along the radial distance. The ratio of the black over the white bars represents the experimental probability of observing a calcium response at a given distance (blue markers, fraction of responding pixels). This probability drops with distance and follows a λ NMDA_GCaMP . Notably, λ NMDA_GCaMP .

Article Snippet: Imaging data for the optical reporter of synaptically released glutamate (iGluSnFr) was acquired on a Nikon A1R MP 2-photon scanning microscope (Nikon) equipped with a BVC-700 (Dagan) amplifier and using WinWCP software (Strathclyde) for current clamp recording.

Techniques: Fluorescence, Expressing, Incubation, Activation Assay

Journal: bioRxiv

Article Title: Optical analysis of the action range of glutamate in the neuropil

doi: 10.1101/2021.02.05.429974

Figure Lengend Snippet: Long iontophoretic glutamate applications were used to minimize the potential buffering effect of membrane-anchored iGluSnfr molecules. (A)Schematic of the experiment (left panel, glutamate iontophoresis pipette in white, iGluSnFR expressing astrocyte in green). Line scans of iGluSnFR fluorescence were obtained in parallel and perpendicular to the CA1 pyramidal layer (left panel, dotted yellow lines) and glutamate was applied for 250 ms (current 10 nA). For analysis and display, the baseline fluorescence intensity (F 0 ) was determined in a 100 ms time window for each x-coordinate (lane) and the ratio F/F 0 was calculated for each lane (middle and right panel, start of iontophoresis illustrated by yellow dotted lines). iGluSnFR saturation was not observed in these experiments and usually required a much stronger glutamate injection (∼100 nA, verified in each experiment). (B) Example of iGluSnFR fluorescence over time during glutamate application (top panel, averaged over entire line scan). The spatial profile of iGluSnFR fluorescence during the last 20 ms of iontophoresis (shaded area in line scans in A ) was analysed to estimate the spatial spread of glutamate in both directions. Spatial spread was quantified by the full width at half maximum of a Gaussian distribution fitted to the fluorescence profiles (lower panel). (C) No statistically significant difference between both directions were observed (n = 10 recordings in 10 different hippocampal slices, parallel and perpendicular line scans always paired, paired Student’s t-test).

Article Snippet: Imaging data for the optical reporter of synaptically released glutamate (iGluSnFr) was acquired on a Nikon A1R MP 2-photon scanning microscope (Nikon) equipped with a BVC-700 (Dagan) amplifier and using WinWCP software (Strathclyde) for current clamp recording.

Techniques: Transferring, Expressing, Fluorescence, Injection

Journal: bioRxiv

Article Title: Optical analysis of the action range of glutamate in the neuropil

doi: 10.1101/2021.02.05.429974

Figure Lengend Snippet: (A)The simulation environment c“calc” ( V. Matveev, A. Sherman, R. S. Zucker, Biophysj. 83, 1368– 1373 (2002) ) (as “spherical symmetry”) was used to replicate the neuropil diffusion models of ( D. A. Rusakov, D. M. Kullmann, J. Neurosci. 18, 3158–3170 (1998), B. Barbour, Journal of Neuroscience. 21, 7969–7984 (2001) ) and to simulate synaptic glutamate release, binding to iGluSnFr and fluorescence activation of iGluSnFr. A gaussian-shaped, fusion pore-like source of glutamate (FWHM 5 nm) was placed at the origin and glutamate was released in a peak-like fashion according to: t * sigma^2 * exp(-sigma*t) (t in ms, sigma=39), following ( D. A. Rusakov, D. M. Kullmann, J. Neurosci. 18, 3158–3170 (1998) ). Glutamate transporters were modeled by omitting the translocation step as fixed glutamate “buffers” at a concentration of 100 µM in the extracellular volume. Omitting the translocation is justified as it is slow and has a negligible effect on free glutamate (not shown, also see B. Barbour, Journal of Neuroscience. 21, 7969– 7984 (2001)). No pre- or postsynaptic structures around the release were modeled as previous studies showed that at the distances considered here (>1000 nm) they have almost no effect on the glutamate concentration. Continuous lines throughout this figure represent calculations in the presence of glutamate “buffers” (k+ = 5e06 /(Ms), k- = 100/s), dashed lines in their absence. An effective glutamate diffusion coefficient D=250µm^2/s was employed to account for the tortuousity of the neuropil similar to ( D. A. Rusakov, D. M. Kullmann, J. Neurosci. 18, 3158–3170 (1998), B. Barbour, Journal of Neuroscience. 21, 7969–7984 (2001) ). The vesicle contained 7000 molecules of glutamate according to recent estimates (see discussion for details). Note that at distances of ≥1500 nm free glutamate concentrations remain below 1 µM, show a slowed rise and peak with at least 1 ms delay. (B) The fraction of iGluSnFr molecules reaching the fluorescent state after synaptic release at 1500 and 2000 nm distance from the release site remained below 0.3%. In other words, classical neuropil diffusion models predict minimal iGluSnFr responses. We modeled iGluSnFr according to ( M. Armbruster, C. G. Dulla, J. S. Diamond, eLife. 9, 10404–26 (2020) ) with 3 states: no glutamate bound, glutamate bound and non-fluorescent and glutamate bound and fluorescent. Rate constants were also taken from that study. (C) Conversion of fractional sniffer activation to fluorescent signals using the fluorescence constants for activated and non-activated iGluSnFr molecules indicated (taken from ( M. Armbruster, C. G. Dulla, J. S. Diamond, eLife. 9, 10404–26 (2020) )). Note that the predicted DF/F iGluSnFr signals remain below 1%, whereas we experimentally determined iGluSnFr amplitudes at a distance of 1500 nm to be ∼5.4% (black horizontal line) following spontaneous, putative quantal, release events. The blue arrow denotes the almost 5-fold differences between the experimental observation and theoretical prediction.

Article Snippet: Imaging data for the optical reporter of synaptically released glutamate (iGluSnFr) was acquired on a Nikon A1R MP 2-photon scanning microscope (Nikon) equipped with a BVC-700 (Dagan) amplifier and using WinWCP software (Strathclyde) for current clamp recording.

Techniques: Diffusion-based Assay, Binding Assay, Fluorescence, Activation Assay, Translocation Assay, Concentration Assay