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Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography and electromyography in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca 2+ and Mg 2+ up to molar equivalents of the phosphodiester/phosphorothioate bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca 2+ alone, Mg 2+ alone, or Ca 2+ and Mg 2+ ) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establish the importance of adding Ca 2+ and Mg 2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides., Competing Interests: A.K. and N.A. are co-founders, on the scientific advisory board, and hold equities of Atalanta Therapeutics. A.K. is a founder of Comanche Pharmaceuticals and on the scientific advisory board of Aldena Therapeutics, AlltRNA, Prime Medicine, and EVOX Therapeutics. N.A. is on the scientific advisory board of the Huntington’s Disease Society of America (HDSA). Select authors hold patents or on patent applications relating to the divalent siRNA and the methods described in this report., (© 2024 The Author(s).)

The largest synaptic input to the sleep-promoting ventrolateral preoptic area (VLPO) [1] arises from the lateral hypothalamus [2], a brain area associated with arousal [3-5]. However, the neurochemical identity of the majority of these VLPO-projecting neurons within the lateral hypothalamus (LH), as well as their function in the arousal network, remains unknown. Herein we describe a population of VLPO-projecting neurons in the LH that express the vesicular GABA transporter (VGAT; a marker for GABA-releasing neurons). In addition to the VLPO, these neurons also project to several other established sleep and arousal nodes, including the tuberomammillary nucleus, ventral periaqueductal gray, and locus coeruleus. Selective and acute chemogenetic activation of LH VGAT(+) neurons was profoundly wake promoting, whereas acute inhibition increased sleep. Because of its direct and massive inputs to the VLPO, this population may play a particularly important role in sleep-wake switching., (Copyright © 2016 Elsevier Ltd. All rights reserved.)