Viral delivery of iGluSnFr in neonates for multi-synapse glutamate imaging in situ
In our previous studies, we introduced optical glutamate sensors in the hippocampal neuropil via stereotaxic viral delivery in young animals [20] or via biolistic transfection in organotypic brain slices [21, 23]. However, brain injections in adults face challenges, such as potential interference with the tissue designated for acute slices, whereas the functional anatomy of organotypic slices might not fully represent that of intact tissue. We, therefore, sought to explore viral transduction in vivo via neonatal intracerebroventricular (ICV) injections (Fig. 1A), aiming at efficient transgene expression in neurons, for up to six weeks post-infection for subsequent ex vivo imaging.
We employed the new generation of AAV-based sensor variant with a relatively high off-rate, AAV9.hSynap.iGluSnFr.WPRE.SV40, but also used the recently described low off-rate sensor variant, SF-iGluSnFr.A184S [24] for comparison. Although AAV9 appeared to penetrate more readily after ICV administration [25] than did AAV2/1, at three to four weeks post-injection both methods provided efficient labelling of Schaffer collateral fragments in s. radiatum (Fig. 1B and 1C). The robust level of expression was maintained for at least six weeks post-infection, which made it suitable for ex vivo slice experiments in young adult animals.
To validate the method, we set out to monitor iGluSnFr fluorescence intensity integrated across the region of interest (incorporating several axonal boutons) during electric stimulation of Schaffer collaterals (five stimuli 50 ms apart; imaging settings as described earlier [20]). For time-lapse imaging, we employed frame-scanning mode providing rapid sampling rate (pixel dwell time 0.5 µs, frame time ~ 25 ms) across the area of interest (256 × 96 pixels, Fig. 1B). The recorded data sets were arranged as T-stacks, consisting of multiple frame scans (typically 35 to 50, depending on the duration of recording). We thus achieved reliable imaging of the dynamics of glutamate release across the sampled tissue fragment (Fig. 1C traces), with clear separation of five responses to individual electric stimuli (Fig. 1C; fEPSP and ΔF/F0 signal traces; one-trial example).
A similar experiment using the slow-unbinding A184S sensor variant (Fig. 1D) revealed robust reporting of stimulus-evoked rises in the iGluSnFr intensity (Fig. 1E). However, this approach did not seem to provide reliable separation between five responses to five individual stimuli at 20 Hz (Fig. 1E; ΔF/F0 trace, one-trial example), thus pointing to the corresponding limitations in temporal resolution.
Multi-synapse imaging of glutamate release at individual axonal boutons
We next asked if the chosen frame-scanning method is sufficiently sensitive to document glutamate release at in individual axonal boutons. We therefore used the recorded image-frame stacks to analyse fluorescence dynamics at small ROIs associated with individual axonal boutons (Fig. 2A). The fluorescence dynamics at individual selected boutons showed that recording sensitivity and signal-noise ratios were sufficient, in principle, to document individual glutamate releases (Fig. 2B; ΔF/F0 traces, four-trial average). For comparison purposes, we recorded a fragment of the same axon (as Fig. 2A) in linescan mode, which provides high temporal resolution (~ 1.45 ms). The fluorescence dynamics thus recorded from three boutons of interest (Fig. 3A, bouton numbers as in Fig. 2A; one-trial example) was qualitatively similar to that obtained in the frame-scanning mode (compare boutons 5–7 in Fig. 2B and Fig. 3B).
Imaging of glutamate release during LTP induction
One of the main advantages of the frame-scan mode, as opposed to various linescan options, is relatively low laser exposure per pixel. Firstly, this lowers the propensity for irreversible photo-damage that may occur in cellular structures under intense laser light. Secondly, it reduces photobleaching of the fluorescent indicator, which has been a key prerequisite for stable longer-term imaging.
We therefore set out to explore this imaging method in an attempt to document changes, if any, of glutamate release during the high-frequency stimulation (HFS)-induced LTP. The classical LTP induction protocol in iGluSnFr-expressing acute slices produced a reliable increase in the fEPSP slope, lasting for over 80 min post-induction (example in Fig. 4A). In the selected areas of s. radiatum, we thus identified groups of candidate axonal boutons that remained firmly in focus during the experiment, to reduce any bias associated with focal drift (Fig. 4B). Unfortunately, the boutons selected based on this mandatory criterion, were not necessarily the boutons showing the best signal-to-noise ratios of their ΔF/F0 responses (as in Fig. 2). Whilst individual boutons displayed, if anything, varied effects of LTP induction on the fluorescence dynamics of iGluSnFR, they appeared to indicate a trend towards the increase in the ΔF/F0 signal amplitude (example in Fig. 4C).
This trend was more prominent when the area-integrated ΔF/F0 signals (as in Fig. 1C, E) were compared (Fig. 4D). To evaluate this effect quantitatively, we carried out paired comparisons, in which the difference was calculated between the average iGluSnFR ΔF/F0 traces recorded at 30 or 90 min post-induction, and those during baseline conditions prior to LTP induction. This analysis indicated a statistically significant increase in the optical glutamate signal amplitude following the induction of LTP (Fig. 4E). Whether this change necessarily indicates a greater amount of evoked glutamate release is discussed below.