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Nanoscale Analytical Methods to gain insight into the Initiation of Short-Term Memory

Periodic Reporting for period 1 - NAMISTMem (Nanoscale Analytical Methods to gain insight into the Initiation of Short-Term Memory)

Berichtszeitraum: 2018-10-24 bis 2020-10-23

Neurons communicate with electrical signals that are converted to chemical signals through exocytosis.
In exocytosis, an electrical signal in the form of an action potential triggers synaptic vesicles filled with neurotransmitter molecules to fuse with the plasma membrane and release these molecules into the synaptic between two neighboring neurons. During the exocytosis, the lipid molecules in the membrane are changed by the simple diffusion of these molecules from the neurotransmitter vesicles to the membrane during release, and this leads to a change in the neurotransmission process to produce a short-term memory (STM). The molecular mechanism that initiates the formation of STM is clearly attributed to the main symptoms in neurodevelopmental and neurodegenerative disorders such as Alzheimer’s disease as well as autism spectrum disorders. These neural disorders showed devastating effects not only on the individual but also society. Despite the vital role in neural disorders, the molecular mechanism of STM initiation is still mostly a mystery. This project aimed to develop novel nano-bioanalytical strategies to explore a molecular paradigm for the initiation of STM based on exocytosis events. Understanding STM initiation at the molecular level requires advanced analytical measurements at the single cell level to monitor individual exocytosis events. This poses an extreme analytical challenge. “NAMISTMem” focused on developing new strategies based on electrodes and electrochemical detection for the quantitative determination of the entire vesicle content and provided a new direction of research to measure cellular changes in presence of chemical endogenous factors (e.g. lipids and zinc) during exocytosis, which correlates these changes to the initial memory formation.

In this project, for the quantitative determination of the entire vesicle content, a microwell array chip (MWAC) was designed and fabricated to trap and detect the entire content of individual vesicles. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in vesicular content analysis and studies of exocytosis as a key part of STM. In another part of the project, using a nano-injection method, we introduced different phospholipids into single cells and used electrochemical methods, single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC), with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process. Our findings provide a crucial piece of data to the hypothesis that lipid heterogeneity and structure might be involved as a regulatory mechanism for exocytotic or synaptic strength in the initial stages of STM. Moreover, developing an analytical approach for understanding the role of zinc ion in STM formation was considered in the project. The initial fabrication of a model ion-selective sensor for this purpose has been performed successfully.
IVIEC, a recently developed in the Ewing group, is a nano-electrochemical technique for in-situ quantification of vesicular content. VIEC is also a similar technique to IVIEC, but instead of in-situ quantification of vesicular content, vesicles are isolated from the cell machinery and then the content is measured. However, in both methods, the vesicles rupture on an electrode surface and it was not clear that if they open towards the electrode or to the other sides. We provided a microwell array electrode to trap the chromaffin vesicles and subsequently their neurotransmitters after rupturing by applying a potential. Our finding showed that the number of neurotransmitter molecules measured in these microwell-arrays was in good agreement with the previously reported results on the regular electrodes. It shows that the entire vesicle content is measured in IVIEC and VIEC; either the vesicle is ruptured towards the electrode or other sides. Thus, these results verify that IVIEC is a robust tool for observing the cellular response of a drug or stimulus on the vesicular content. The obtained result was published as a scientific article in an American Chemical Society publication:
Ranjbari, E., Taleat, Z., Mapar, M., Aref, M., Dunevall, J., & Ewing, A. (2020). Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays. Analytical Chemistry, 92(16), 11325-11331.

Intracellular lipid injection, as a promising strategy, was developed to in-situ manipulate the lipidic structure of not only the cell membrane inner leaflet but the outer leaflet of the vesicles. We injected/delivered different phospholipids possessing different intrinsic curvature into chromaffin single cells using a nanopipette and monitored the effect of so-called intracellular incubation of phospholipids on the exocytosis release process and also the vesicular content using SCA and IVIEC techniques, respectively. This work provides new insights into the mechanism of synaptic plasticity. Our exocytotic analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways and also provides new evidence for a critical role of the lipid localization in modulating the release process. The obtained result was published as a scientific article in the Royal Society of Chemistry
Aref, M., Ranjbari, E., Romiani, A., & Ewing, A. G. (2020). Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry. Chemical Science, 11(43), 11869-11876.

A new methodology to study zinc’s effect on exocytosis and memory formation was proposed. In this part of the project, two electrochemical techniques (potentiometry and amperometry at single cell level) were considered to combine. To develop and apply the potentiometric sensor for in-vivo zinc measurement and study the role of zinc in STM formation, fabrication of a model potentiometric sensor, the pH sensor, was necessary. This model sensor has been successfully developed. However, before moving to the zinc sensor development, the project reached its 24th month. The initial results of this sensor for intracellular monitoring will be reported in a manuscript.
As phospholipids are the most abundant constituent of cellular membranes and it is accepted that an important part of drug action in the brain might be alteration of lipid distribution, our data could be beneficial for the development of drugs and novel pharmacological tools. In addition, these findings could offer a sensitive analytical modality to understand how intracellular changes in lipid species, both in the inner membrane leaflet and the intracellular vesicles might be involved in relation to diseases involving memory and cognition (e.g. Alzheimer’s disease and autism).
Our finding suggests that
Lipid injection into a single cell and effect of lipids on cellular membrane