Tengolics, A. J. | bioRxiv Science

bioRxiv2025

High-throughput measurements of neuronal activity in single human iPSC-derived glutamate neurons

Induced pluripotent stem cell (iPSC)-derived neurons provide a promising platform for studying neuronal function and modeling central nervous system (CNS) diseases. However, functional analysis of large populations of iPSC-derived neurons has been challenging. Here, we developed a high throughput strategy targeting N-methyl-D-aspartate receptors (NMDA-R) to enhance neuronal activity and reveal functional phenotypes in human iPSC-induced glutamatergic neurons (iGlut). Using a genetically encoded calcium indicator (GCaMP8f), we first demonstrate that using artificial cerebrospinal fluid (ACSF) lacking Mg2+ (Mg2+-free) significantly increases neuronal firing, and that firing is enhanced by a potentiator (glycine) but inhibited by the NMDA-R antagonist AP-V. Similarly, multi-electrode array (MEA) recordings also show robust firing in Mg2+-free ACSF. Lastly, single-cell patch-clamp electrophysiology confirms the high firing rates in Mg2+-free ACSF across multiple iPSC donor lines and also reveals iPSC donor-specific tonic and bursting firing phenotypes. This new methodology provides a scalable, high-throughput method to study neuronal activity in iGlut neurons while preserving single-cell resolution. The strategy also reveals different functional phenotypes, enabling detailed characterization of iGlut neurons in diverse applications such as CNS disease modeling and drug screening. These findings establish a versatile framework for future studies of neuronal network dynamics and individual excitability in iPSC-derived neuronal cultures.

neuroscience↗

bioRxiv2022

Traumatic brain injury induced microglial and Caspase3 activation in the retina

Traumatic brain injury (TBI) is among the main causes of sudden death after head trauma. These injuries can result in severe degeneration and neuronal cell death in the CNS, including the retina which is a crucial part of the brain responsible for perceiving and transmitting visual information. The long-term effects of mild-repetitive TBI (rmTBI) are far less studied thus far, even though damages induced by repetitive injuries occurring in the brain are more common, especially amongst athletes. rmTBI can also have a detrimental effect on the retina and the pathophysiology of these injuries are likely to differ from the severe TBI (sTBI) retinal injury. Here we showed how rmTBI and sTBI can dissimilarly affect the retina. Our results indicate an increase in the number of activated microglial cells and Caspase3-positive cells in the retina in both traumatic models, suggesting a rise in the level of inflammation and cell death after TBI. The pattern of microglial activation appears evenly distributed and widespread but differs amongst the various retinal layers. sTBI induced microgial activation in both the superficial and deep retinal layers. In contrast to sTBI, no significant change occurred following the repetitive mild injury in the superficial layer, only the deep layer (spanning from the inner nuclear layer to the outer plexiform layer) shows microglial activation. This difference suggests that alternate response mechanisms play a role in the case of the different TBI incidents. The Caspase3 activation pattern showed a uniform increase in both the superficial and deep layers of the retina. This suggests a different action in the course of the disease in sTBI and rmTBI models and points to the need for new diagnostic procedures. Our present results suggest that the retina might serve as such a model of head injuries since the retinal tissue reacts to both forms of TBI and is the most accessible part of the human brain.

neuroscience↗