Biphasic response of human iPSC-derived neural network activity following exposure to a sarin-surrogate nerve agent.

Organophosphorus nerve agents (OPNA) are hazardous environmental exposures to the civilian population and have been historically weaponized as chemical warfare agents (CWA). OPNA exposure can lead to several neurological, sensory, and motor symptoms that can manifest into chronic neurological illnesses later in life. There is still a large need for technological advancement to better understand changes in brain function following OPNA exposure. The human-relevant in vitro multi-electrode array (MEA) system, which combines the MEA technology with human stem cell technology, has the potential to monitor the acute, sub-chronic, and chronic consequences of OPNA exposure on brain activity. However, the application of this system to assess OPNA hazards and risks to human brain function remains to be investigated. In a concentration-response study, we have employed a human-relevant MEA system to monitor and detect changes in the electrical activity of engineered neural networks to increasing concentrations of the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). We report a biphasic response in the spiking (but not bursting) activity of neurons exposed to low (i.e., 0.4 and 4 µM) versus high concentrations (i.e., 40 and 100 µM) of NIMP, which was monitored during the exposure period and up to 6 days post-exposure. Regardless of the NIMP concentration, at a network level, communication or coordination of neuronal activity decreased as early as 60 min and persisted at 24 h of NIMP exposure. Once NIMP was removed, coordinated activity was no different than control (0 µM of NIMP). Interestingly, only in the high concentration of NIMP did coordination of activity at a network level begin to decrease again at 2 days post-exposure and persisted on day 6 post-exposure. Notably, cell viability was not affected during or after NIMP exposure. Also, while the catalytic activity of AChE decreased during NIMP exposure, its activity recovered once NIMP was removed. Gene expression analysis suggests that human iPSC-derived neurons and primary human astrocytes resulted in altered genes related to the cell's interaction with the extracellular environment, its intracellular calcium signaling pathways, and inflammation, which could have contributed to how neurons communicated at a network level.

Corrigendum: Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

[This corrects the article DOI: 10.3389/fncel.2023.1287089.].

Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures.

Fentanyl is one of the most common opioid analgesics administered to patients undergoing surgery or for chronic pain management. While the side effects of chronic fentanyl abuse are recognized (e.g., addiction, tolerance, impairment of cognitive functions, and inhibit nociception, arousal, and respiration), it remains poorly understood what and how changes in brain activity from chronic fentanyl use influences the respective behavioral outcome. Here, we examined the functional and molecular changes to cortical neural network activity following sub-chronic exposure to two fentanyl concentrations, a low (0.01 µM) and high (10 µM) dose. Primary rat co-cultures, containing cortical neurons, astrocytes, and oligodendrocyte precursor cells, were seeded in wells on either a 6-well multi-electrode array (MEA, for electrophysiology) or a 96-well tissue culture plate (for serial endpoint bulk RNA sequencing analysis). Once networks matured (at 28 days in vitro), co-cultures were treated with 0.01 or 10 µM of fentanyl for 4 days and monitored daily. Only high dose exposure to fentanyl resulted in a decline in features of spiking and bursting activity as early as 30 min post-exposure and sustained for 4 days in cultures. Transcriptomic analysis of the complex cultures after 4 days of fentanyl exposure revealed that both the low and high dose induced gene expression changes involved in synaptic transmission, inflammation, and organization of the extracellular matrix. Collectively, the findings of this in vitro study suggest that while neuroadaptive changes to neural network activity at a systems level was detected only at the high dose of fentanyl, transcriptomic changes were also detected at the low dose conditions, suggesting that fentanyl rapidly elicits changes in plasticity.

Interleukin-6 mediates delirium-like phenotypes in a murine model of urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) is frequently implicated as a precipitant of delirium, which refers to an acute confusional state that is associated with high mortality, increased length of stay, and long-term cognitive decline. The pathogenesis of delirium is thought to involve cytokine-mediated neuronal dysfunction of the frontal cortex and hippocampus. We hypothesized that systemic IL-6 inhibition would mitigate delirium-like phenotypes in a mouse model of UTI. METHODS: C57/BL6 mice were randomized to either: (1) non-UTI control, (2) UTI, and (3) UTI + anti-IL-6 antibody. UTI was induced by transurethral inoculation of 1 × 108 Escherichia coli. Frontal cortex and hippocampus-mediated behaviors were evaluated using functional testing and corresponding structural changes were evaluated via quantification of neuronal cleaved caspase-3 (CC3) by immunohistochemistry and western blot. IL-6 in the brain and plasma were evaluated using immunohistochemistry, ELISA, and RT-PCR. RESULTS: Compared to non-UTI control mice, mice with UTI demonstrated significantly greater impairments in frontal and hippocampus-mediated behaviors, specifically increased thigmotaxis in Open Field (p < 0.05) and reduced spontaneous alternations in Y-maze (p < 0.01), while treatment of UTI mice with systemic anti-IL-6 fully reversed these functional impairments. These behavioral impairments correlated with frontal and hippocampal neuronal CC3 changes, with significantly increased frontal and hippocampal CC3 in UTI mice compared to non-UTI controls (p < 0.0001), and full reversal of UTI-induced CC3 neuronal changes following treatment with systemic anti-IL-6 antibody (p < 0.0001). Plasma IL-6 was significantly elevated in UTI mice compared to non-UTI controls (p < 0.01) and there were positive and significant correlations between plasma IL-6 and frontal CC3 (r2 = 0.5087/p = 0.0028) and frontal IL-6 and CC3 (r2 = 0.2653, p < 0.0001). CONCLUSIONS: These data provide evidence for a role for IL-6 in mediating delirium-like phenotypes in a mouse model of UTI. These findings provide pre-clinical justification for clinical investigations of IL-6 inhibitors to treat UTI-induced delirium.