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Nano-physiology of small glutamatergic axon terminals

Periodic Reporting for period 2 - nanoAXON (Nano-physiology of small glutamatergic axon terminals)

Reporting period: 2019-10-01 to 2021-03-31

Publications were not expected in the first reporting period that arose directly from this project. The progress that we made in the first half makes me confident that, as planned, we will publish 3-5 original research papers and one methodological paper in leading international journals at the end of the ERC-CoG support. From these, the manuscript of the methodological paper is completed and will be submitted after all co-authors approve the final version. Furthermore, currently we are preparing two publications concerning Aim1a, 1b and 1c. In addition to the planned tasks we contributed to a project that was started earlier and will help the completion of the ERC-CoG because it allowed us to acquire new methodological approaches. This work was published (Olah et al. eLife).
The major goals of the first half of the project were to establish new methods and collection of data. Specifically, experiments that address the size-dependence of axonal signaling were completed and experiments that measure the pathway-specific signaling are nearing completion.

Aim 1a: In this task we used whole bouton recordings from small axon terminals of mossy fiber, whose size matches typical cortical axons. Much of our knowledge about axonal signaling came from the unusually large boutons of the mossy fibers and our new data is a direct comparison of the unusually large but well-known and the small but more typical axonal boutons. We are preparing two manuscripts from these results, which suggest that in spite of their different biophysical properties, electrical signaling in small and large axon terminals along the same axons is uniform. A major advancement within these aim was the establishment of axonal signal measurement using voltage-sensitive dyes. For this we acquired and tested new specialized equipment and new experts were hired (see below). The advantage of voltage-sensitive dye imaging is that we can simultaneously monitor action potential waveforms at multiple locations of the same axon. Thus, the combination of direct electrophysiology and voltage imaging provide unprecedented insight into signaling within single axons.
Furthermore, we performed simulations about the constraints that small pipettes impose on small axonal recordings and we built a complex computational model that enables us and others to correct these errors and obtain the native action potential waveforms even from the smallest axonal structures. This methodological approach resulted in the third manuscript that will be submitted within this 2020. Additionally, as planned we are performing similar direct recordings from individual axons in the dentate that arrive from the major glutamatergic inputs, including SuM nucleus, mossy cells and two types of perforant pathways.
Aim 1b: This aim was designed to reveal the underlying ionic conductances of action potentials in small axonal terminals. As proposed in the original grant application we used outside-out patch clamp experiments to directly measure these conductance and then implement these into biologically realistic computational models for verification. Our results show that small mossy fiber axonal bouton achieve similar action potentials as large boutons by a higher ratio of potassium and sodium channels. These findings are supporting one of the manuscript mentioned above.
The goal of Aim 1c is to measure axonal calcium influx evoked by the native action potentials of the different boutons. For this aim we were able to directly measure calcium currents from different types of mossy fiber axon terminals. Please note that at the time of the submission of the grant application we proposed a more indirect measurement for this aim, but due to further improvements we were able to collect the necessary data with the direct measurements of axonal calcium current, which can be easily interpreted. Furthermore, the stable axonal calcium current measurements allowed to dissect the underlying types of calcium channels using specific pharmacological tools. These results are part of the second manuscript that is being prepared.

Aim 2a: A new approach that we implemented in the first period of the supports adds novel and more detailed insight into this question. Shortly, we can test large number of potential postsynaptic partners and map the synaptic targets of individual PP, mossy cell and SuM axons within the DG not only by anatomically but also functionally. The adaptation of this technique will provide more efficient and detailed answers to the questions of the proposal.
One dedicated person is finetuning the anatomical procedures that allow us to precisely identify individual axons and postsynaptic cells. This conventional morphological experiments needs large amount of control experiments for antibody testing for which an expert has been employed for the project (see below). Furthermore, we are exploring the possibility of using virus-vectors that allows a manipulation of specific types of axons more precisely by expression of proteins under the control of new promoter sequences. We are also considering whether measurement of axonal RNA contents is possible and suitable for axonal identification and/or revealing the underlying molecular mechanisms (see below Olah et al. 2020).
Aim2b: Impact of single axons on the dentate circuit. Emergence of new voltage-sensitive protein sensors allowed us to refine the experimental arrangement of this aim, which will generate data more efficiently. Specifically, some of the new voltage-sensors is sufficiently sensitive for detecting subthreshold membrane potential changes in a well-defined subset of cells, in our project in dentate gyrus neurons. With this new approach, which we call Voltron pair recordings from the name of the voltage sensor, we can monitor several postsynaptic cells simultaneously (instead of one with conventional paired recordings), while we stimulate single glutamatergic axons in the dentate using direct recordings. I am convinced that the combination of our methodological advantages in axonal recording with the new voltage imaging technique will not only efficiently address the questions of the project but will provide a unique cutting edge methods for us.
All arms of the project is progressing as planned. We have collected sufficient data that addresses whether and how small axonal terminals are different from the better studied but unusually large axon terminals. We are addressing two basic science questions with these results in two manuscripts that are being prepared. Furthermore, we created a simulation environment which allows for the corrections of bias caused by the recording instruments in small axonal membranes. Thus, we can predict how native axonal electrical signal would look if the recording instruments were not present. We will submit the completed manuscript about this approach within the next month.
As described in other sections of the report the project received a boost by the implementation of the emerging experimental tools that enable us to more precise and efficiently map the impact of single glutamatergic fibers arriving to and from the dentate gyrus region. Thus we will determine in detail how different incoming information generate the unique neuronal output of this brain region.
Furthermore, in addition to the planned tasks we published a paper that started earlier and will help the completion of the ERC (Olah et al. 2020 eLife).