A high-density multi-electrode platform examining the effects of radiation on in vitro cortical networks

Abstract

Radiation therapy and stereotactic radiosurgery are common treatments for brain malignancies. However, the impact of radiation on underlying neuronal circuits is poorly understood. In the prefrontal cortex (PFC), neurons communicate via action potentials that control cognitive processes, thus it is important to understand the impact of radiation on these circuits. Here we present a novel protocol to investigate the effect of radiation on the activity and survival of PFC networks in vitro. Escalating doses of radiation were applied to PFC slices using a robotic radiosurgery platform at a standard dose rate of 10 Gy/min. High-density multielectrode array recordings of radiated slices were collected to capture extracellular activity across 4,096 channels. Radiated slices showed an increase in firing rate, functional connectivity, and complexity. Graph-theoretic measures of functional connectivity were altered following radiation. These results were compared to pharmacologically induced epileptic slices where neural complexity was markedly elevated, and functional connections were strong but remained spatially focused. Finally, propidium iodide staining revealed a dose-dependent effect of radiation on apoptosis. These findings provide a novel assay to investigate the impacts of clinically relevant doses of radiation on brain circuits and highlight the acute effects of escalating radiation doses on PFC neurons.

Keywords: Complexity; Functional connectivity; Multielectrode array; Neuronal activity; Prefrontal cortex; Radiation.

Fig. 1

Neuronal activity in populations of prefrontal cortical neurons. (A) Left, example of PFC slice placement on the hd-MEA. Right, multiunit spike (identified by an asterisk “*”) extracted from voltage deflections identified at an individual channel. (B) Mean firing rate of prefrontal cortical neurons across various conditions. C = control slices for the radiation treatment; BL = baseline in the PE condition; 20–100 Gy = radiation conditions; EP = epileptiform activity. *indicates statistical significance at p < 0.01. (C) Rasters of activity show spikes across whole populations of neurons following graded doses of radiation or perfusion of pro-epileptiform solution. Peri-stimulus time histogram underneath each raster shows summated activity using a 500 ms rolling window.

Fig. 2

Functional connectivity of cortical networks. Radiation (A). Perfusion of a pro-epileptiform (PE) solution (B). Mean strength (C) and number of functional connections exceeding a pre-defined threshold (D). Asterisks (“*”) indicate significance at p < 0.01 (Wilcoxon rank sum test).

Fig. 3

Graph-theoretic properties of functional connectivity. Modularity (A), global and local efficiency (B), assortativity (C), and transitivity (D) were computed across experimental conditions. A cartoon illustration of each measure is shown above figures.

Fig. 4

Alterations in global synchronization. (A) Synchronization is computed by the correlation between spikes at individual channels (top) and the PSTH obtained by summing all neurons in the population after averaging with a rolling temporal window. (B-C) Mean synchronization across conditions using a rolling window of 500 ms or 250 ms.

Fig. 5

Neuronal complexity of cortical networks. Participation ratio (PR) in radiated slices (A). PR in slices treated with PE solution (B). Distribution of eigenvalues across experimental conditions (C).

Fig. 6

Propidium iodide (PI) staining of prefrontal slices following radiation. (A) Representative samples showing a graded increase in PI intensity with radiation dose. Arrow shows clustering of PI intensity at 100 Gy. Left panel shows approximate location of secondary motor cortex (M2), Prelimbic cortex (PrL), medial orbital cortex (MO), ventral orbital cortex (VO), lateral orbital cortex (LO), and dorsolateral orbital cortex (DLO), (B) Mean PI intensity (“a.u.”: arbitrary units).

Fig. 7

Illustration of the protocol employed to perform radiation, record neural activity, and apply PI staining to prefrontal cortical slices. A comparison of activity under radiation and PE treatment was performed.