Characterization of 4-Aminopyridine-induced network plasticity in rat cortical and human iPSC-derived neuronal cultures

Abstract

Event Abstract Back to Event Characterization of 4-Aminopyridine-induced network plasticity in human iPSC-derived neuronal cultures Gaye Tanriöver1* and Katja Nieweg1 1 Phillips University, Institute of Pharmacology and Clinical Pharmacy, Germany Motivation Homeostatic network plasticity is a key regulatory mechanism for fine-tuning network activity. 4-aminopyridine (4-AP), which is commonly used to model epilepsy in vitro and in vivo has recently been described to trigger homeostatic plasticity in cultures of mouse hippocampal neurons [1]. The inhibition of several types of K+ channels (D-type and A-type K+ currents and a fraction of delayed rectifier currents) by 4-AP leads to prolonged action potentials and increased neurotransmitter release [2]. This induces hyperactivity, which is counteracted by mechanisms leading to decreased neuronal excitability. Human induced pluripotent stem cell (hiPSC)-derived neurons have been proposed to be a highly valuable in vitro model to be used in neurotoxicology, drug screening and regenerative medicine. Since spontaneous synchronous network activity develops in hiPSC-derived neuronal networks, our aim was to study the capacity of hiPSC-derived neuronal networks to exhibit homeostatic plasticity in response to 4-AP. Material and Methods hiPSCs were differentiated to cortical neurons as described previously [3]. Fifty thousand cells were plated after enrichment of neurons by immunopurification. After long-term (2-3 months) culture of the neurons for maturation, networks were transferred on the PO and laminin-coated 60-electrode multi-electrode arrays (Multichannel Systems, 200 µm electrode distance, 30µm electrode diameter) (MEAs) for recording. Calcium imaging was performed using Fura-2-AM ratiometric dye to identify single cell activity. Cells were loaded with 3µM Fura-2 AM for 20 min at 37°C in HEPES buffer. Fluorescence intensities at the two wavelengths (F340 and F380) were recorded separately and combined (fluorescence ratio: r=F340/F380) after background subtraction. Cells were imaged before and during 4-AP treatment as well as after removal of 4-AP. Neuronal networks with spontaneous network activity were incubated with 4-AP (100 µM) in order to trigger hyperactivity and measured during 4 days of 4-AP treatment as well as post-washout to monitor homeostatic plasticity. Results We modified our previously published [3] protocol for immunopanning of human iPSC derived neurons in order to permit differentiation of astrocytes from persisting neural precursor cells. Thus, we were able to generate mature, spontaneously active cortical like astrocyte-neuronal networks, shown by the presence of synchronous calcium transients around two months after immunoisolation. Analysis of network maturation was performed by culturing on 60-electrode MEAs. Single spikes and spike trains could be detected long before the occurrence of synchronous network bursts, which appeared two to three months after immunoisolation. To test, whether 4-AP triggered hyperactivity can induce homeostatic plasticity in human iPSC derived neuronal networks as it has been described for mouse hippocampal neurons [1], we treated human neurons of different maturational stages with 100µM 4-AP for 2-4 days and analyzed spontaneous network activity by MEA and calcium imaging. Mature, bursting human networks responded to 4-AP treatment with increased spiking and bursting rates (Fig. 1). Within the first 24h of treatment, mechanisms of homeostatic plasticity lead to downregulation of the activity close to baseline levels. One day after 4-AP removal, we observed that the overall network activity was strongly reduced even below baseline levels. The activity slowly recovered during the following days. Calcium imaging, which permits analyzing the activity of single cells within a network revealed similar results in mature human networks. However, immature human cultures without synchronous bursting activity did not respond to 4-AP and thus homeostatic plasticity could not be triggered in these cultures. Discussion 4-AP treatment of dissociated mouse hippocampal neurons cultured on MEAs was described, as a model to study homeostatic network plasticity by Pozzi et al. [1]. They showed that 4-AP induced hyperactivity lead to downregulation of neuronal excitability in mouse cultures. In our study, we applied this model to hiPSC derived neuronal networks and could show, that the maturational status of the network is a crucial parameter. Only spontaneously bursting cultures were able to respond in the expected manner. Whether downregulation of overall network activity occurs at the level of neuronal excitability or by synaptic downscaling will be analyzed by patch clamp recording. Conclusion In our study, we could show that mature hiPSC-derived neuronal networks cultured on MEAs are a valuable model to investigate mechanisms of homeostatic network plasticity in vitro. Since homeostatic plasticity is impaired in many psychiatric disorders and also at early stages of neurodegenerative diseases, a combination of patient derived iPSCs and the 4-AP plasticity model could serve as an innovative platform for drug screening.