Abstract
Motor neurons are the site of action for several neurological disorders and paralytic toxins, with cell bodies located in the ventral horn (VH) of the spinal cord along with interneurons and support cells. Microelectrode arrays (MEAs) have emerged as a high content assay platform for mechanistic studies and drug discovery. Here, we explored the spontaneous and evoked electrical activity of VH cultures derived from embryonic mouse spinal cord on multi-well plates of MEAs. Primary VH cultures from embryonic day 15–16 mice were characterized by expression of choline acetyltransferase (ChAT) by immunocytochemistry. Well resolved, all-or-nothing spontaneous spikes with profiles consistent with extracellular action potentials were observed after 3 days in vitro, persisting with consistent firing rates until at least day in vitro 19. The majority of the spontaneous activity consisted of tonic firing interspersed with coordinated bursting across the network. After 5 days in vitro, spike activity was readily evoked by voltage pulses where a minimum amplitude and duration required for excitation was 300 mV and 100 μs/phase, respectively. We characterized the sensitivity of spontaneous and evoked activity to a host of pharmacological agents including AP5, CNQX, strychnine, ω-agatoxin IVA, and botulinum neurotoxin serotype A (BoNT/A). These experiments revealed sensitivity of the cultured VH to both agonist and antagonist compounds in a manner consistent with mature tissue derived from slices. In the case of BoNT/A, we also demonstrated intoxication persistence over an 18-day period, followed by partial intoxication recovery induced by N- and P/Q-type calcium channel agonist GV-58. In total, our findings suggest that VH cultures on multi-well MEA plates may represent a moderate throughput, high content assay for performing mechanistic studies and for screening potential therapeutics pertaining to paralytic toxins and neurological disorders.
Highlights
Spinal motor neurons (MNs) constitute the final common neural pathway for the control of movement
74.5% were found to be choline acetyltransferase (ChAT) positive (Figure 1F). These data indicate that our dissection and culturing protocols led to successful culturing of MNs isolated from ventral spinal cord and suggest that the resulting extracellular recordings should reflect contributions from MNs as well as other ventral spinal neurons; most likely inhibitory interneurons
We have quantitatively characterized the emergence of spontaneous single-unit as well as network activity in a ventral horn (VH) culture preparation using Microelectrode arrays (MEAs), and that network activity may be evoked by electrical stimulation
Summary
Spinal motor neurons (MNs) constitute the final common neural pathway for the control of movement. Activity from Ventral Horn Cultures neurotoxins act on MNs at the synapse to suppress neurotransmitter release, producing paralysis during poisoning (Burgen et al, 1948; Parsons et al, 1966). Given their importance, numerous screening and mechanistic studies have made use of primary MNs (Kuo et al, 2004), tumorderived MN-like cells (Maier et al, 2013), or stem cell-derived MNs (Sances et al, 2016) in assays often relying on cytotoxicity, neurite extension, or DNA damage as an endpoint. Our findings suggest that VH cultures on multi-well plates of MEAs may enable a stable high content platform for screening potential therapeutics and mechanistic studies pertaining to paralytic toxins and neurological disorders
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