A MULTIMODAL PIPELINE FOR <EM>EX-VIVO </EM>HUMAN BRAIN SLICES: INTEGRATING HD-MEA, 2-PHOTON IMAGING, AND PATCH-CLAMP ELECTROPHYSIOLOGY

Most of the clinical and preclinical research relies on animal models, while species-specific human models are essential to bridge the translational gap which remain a major bottleneck in neuroscience research (Seyahn, 2019). Here we present an experimental protocol to perform electrophysiological studies on acute human brain slices. We utilized a multimodal approach integrating high-density microelectrode arrays (HD-MEAs) for extracellular recordings, 2-photon microscopy (2PM) for calcium imaging, and patch-clamp for somatic intracellular recordings. Human samples, collected from temporal lobe therapeutic surgeries of drug-resistant epileptic patients are incubated in a modified aCSF, called NMDG aCSF, during transportation and slicing. After recovery, slices were alternatively placed in the chamber of a 2P microscope, on the inner surface of a 3-dimensional HD-MEA or used for whole-cell patch-clamp experiments. HD-MEA and 2P experiments were performed while bathing the slices with control aCSF as well as two epileptogenic conditions a) 0 mM Mg²⁺ extracellular concentration combined with Gabazine (10 µM), or b) high K⁺ concentration (8 mM) combined with 4-AP (100 µM). No interictal-like events were observed under control aCSF, while their occurrence was prominent under the altered extracellular solution. Intrinsic excitability was further studied with intracellular recordings, exclusively using control aCSF solution complemented with ionotropic excitatory and inhibitory synaptic blockers. Our combined dataset suggests that HD-MEA and calcium imaging effectively capture microcircuit synchronization, while single-cell recordings clarify the role of single neurons in these dynamics. This approach will advance our understanding of network dynamics in drug-resistant epilepsy, paving the way for more effective therapeutic strategies.