582 - Age-Dependent Network Effects of Cannabidiol in Neonatal Seizure Circuits Revealed by Microelectrode Array Recordings

Publication Number: 3563.582

Fahad Iqbal, University of Calgary, Calgary, AB, Canada; Umar Zaidi, University of Calgary, Calgary, AB, Canada; Naweed I.. Syed, University of Calgary, Calgary, AB, Canada; Kamran Yusuf, Kamran Yusuf, Department of Pediatrics, University of Calgary, Calgary, AB, Canada

Background: Current anti-seizure medications (ASMs) are limited by safety concerns, as broad inhibitory mechanisms can interfere with normal brain development. Cannabidiol (CBD), a recently approved ASM for pediatric epilepsies, shows promise in several seizure syndromes but has not been systematically evaluated in neonates. Given the vulnerability of the developing brain, understanding how CBD modulates seizure-like activity (SLA) and interacts with neonatal circuitry is essential to assess its safety and potential long-term consequences.

Objective: To characterize the effects of CBD on SLA in neonatal hippocampal circuits and identify receptor-specific mechanisms.

Design/Methods: Hippocampal slices (5≥ biological replicates per condition) from neonatal (P0-P5) Sprague Dawley rats were interfaced with 3D microelectrode arrays. SLA was induced using magnesium-free artificial cerebrospinal fluid, and network excitability, burst dynamics, and synchrony were quantified using a custom-developed analysis pipeline. Two potential molecular pathways targets (GP55, a G-protein coupled receptor, and nAChRs) were assessed for their role in the mechanism of action for CBD. Long-term cell viability and synaptic network assembly were assessed in primary hippocampal cultures using fluorescence imaging.

Results: CBD produced opposing age-dependent effects on hippocampal network activity. In neonatal slices, CBD increased burst frequency and network synchrony, generating large depolarizing events. These effects were unexpected, as they contrast with the known inhibitory actions of CBD in mature brain tissue, and may point to GABA-mediated depolarizing waves in the neonatal tissue. Blocking GP55 and nAChRs differentially impacted neuronal activity in the presence of CBD, highlighting the functional role of these receptors in the mechanism of CBD.

In adult hippocampal circuits, CBD instead reduced seizure-like discharges and restored baseline activity, indicating a shift toward inhibition with maturation, as is seen clinically. Immunofluorescence imaging of cultured neurons confirmed that CBD-treated networks exhibited altered synaptic organization, suggesting functional network remodeling.

Conclusion(s): Contrary to mature brain networks, CBD increases excitability in neonatal hippocampal networks through convergent, receptor-mediated mechanisms, which warrants further investigation before use at this developmental stage. These effects lead to long-term changes in synaptic network assembly and underscore the importance of developmental-stage-specific evaluation of ASMs, particularly in the context of early postnatal development.

Multimodal Analysis of Neonatal Seizure Networks Using Microelectrode Arrays and Synaptic Imaging
(A) Representative image showing a neonatal hippocampal brain slice interfaced on a 3D microelectrode array (MEA). Regions of interest are labeled. Depending on the experiment, the hippocampus may be positioned to capture activity patterns from regions of interest. (B) Representative electrode array panel undergoing synchronized, high-amplitude compound field potentials (CFPs), separated by tonic spike trains in some channels. These electrodes are independent to the image in (A). (C) Close-up of a channel in the dentate gyrus region, showing tonic spike trains leading up to a large-amplitude CFP. (D) Snapshot of filtered raw data and spike waveform cutouts from a recording of a mature neuronal culture on a 2D MEA. (E) Sample of waveform comparison between baseline and zero-Mg2+-induced SLA in dissociated neuronal cultures, showcasing unique SLA characteristics in this approach. (F) Representative pictures of neurons labeled with DAPI for nuclear staining, PSD-95 (Green), and GluR1 (Red) which indicate the presence of glutamatergic synapses at early developmental stages.