Thesis Project Form

Ischemic injury in the brain causes significant disruptions in neuronal ion homeostasis due to oxygen and glucose deprivation (OGD). The failure of the Na?/K?-ATPase pump leads to intracellular accumulation of Na? and Ca²?, osmotic imbalance, and sustained membrane depolarization. This chain of events results in uncontrolled glutamate release and subsequent excitotoxicity, which are major contributors to neuronal death during ischemia.
To address this issue, the project proposes using microelectrode arrays (MEAs) to deliver patterned electrical stimulation protocols designed to induce long-term depression (LTD) in mixed human iPSC-derived 3D neuronal and astrocyte cultures. LTD is a form of synaptic plasticity characterized by a lasting decrease in synaptic strength. It can be mediated by presynaptic mechanisms, such as reduced glutamate release, and postsynaptic mechanisms, including diminished AMPA receptor conductance and receptor internalization. By exploiting these mechanisms, we hypothesize that inducing LTD during and after ischemic insult models can reduce network hyperexcitability, stabilize neuronal activity, and ultimately lessen excitotoxic damage.

The objective of this thesis is to investigate whether LTD-inducing patterned stimulation can serve as a neuroprotective strategy. The project is structured in three main tasks:
1) Developing a stable modality for the induction of ischemia in vitro.
a. Exploiting two experimental set-ups, one for MEA-based electrophysiology and one for calcium imaging acquisitions.
b. Optimizing an in vitro model with human mixed neuronal/astrocyte culture.
c. Testing two types of ischemia: (i) oxygen-glucose deprivation (OGD); (ii)NMDA treatment.
2) Investigating the impact of ischemia
a. Evaluating cell viability and cell death pathways through biochemical assays (i.e. LDH release, caspase-3/7 activation).
b. Recordings from MEAs to provide quantitative data on firing rate, burst frequency, synchrony, and overall network homeostasis
c. Calcium imaging using the ratio-metric dye FuraRed for precise quantification of cytosolic Ca²? dynamics.
3) Inducing LTD
a. Optimizing LTD induction protocols using MEAs on control neuronal/astrocyte cultures by fine-tuning stimulation parameters.
b. Validation through MEA recordings, by assessing changes through typical electrophysiological features.
c. Applicating the LTD protocol to cultures during and after OGD or NMDA treatment to evaluate its effect on cellular responses under excitotoxic stress.

The student will work in a Laboratory environment and acquire expertise in the application of MEA-based electrophysiology as well as Calcium imaging within in vitro ischemia models. Training will also include the development and refinement of analytical pipelines for extracellular recording datasets and the use of dedicated software for the analysis of calcium imaging data.