Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5β-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5β-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5β-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5β-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

Charles University 3rd Faculty of Medicine Ruska 87 100 00 Prague 10 Czech Republic

CZ OPENSCREEN Institute of Molecular Genetics CAS Videnska 1083 142 20 Prague 4 Czech Republic

Institute of Biotechnology CAS Prumyslova 595 252 50 Vestec Czech Republic

Institute of Endocrinology Narodni 8 116 94 Prague 1 Czech Republic

Institute of Molecular Genetics CAS Videnska 1083 142 20 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry CAS Flemingovo nam 2 166 10 Prague 2 Czech Republic

Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic

IT4Innovations National Supercomputing Center Studentska 6231 1B 708 00 Ostrava Poruba Czech Republic

Laboratory of Cellular Neurophysiology Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic

National Institute of Mental Health Topolova 748 250 67 Klecany Czech Republic

Zobrazit více v PubMed

Baulieu E. E. Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Recent Prog. Horm. Res. 1997, 52, 1–32. PubMed

Belelli D.; Lambert J. J. Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat. Rev. Neurosci. 2005, 6, 565–575. 10.1038/nrn1703. PubMed DOI

Korinek M.; Kapras V.; Vyklicky V.; Adamusova E.; Borovska J.; Vales K.; Stuchlik A.; Horak M.; Chodounska H.; Vyklicky L. Neurosteroid modulation of N-methyl-D-aspartate receptors: molecular mechanism and behavioral effects. Steroids 2011, 76, 1409–1418. 10.1016/j.steroids.2011.09.002. PubMed DOI

Mellon S. H.; Griffin L. D.; Compagnone N. A. Biosynthesis and action of neurosteroids. Brain Res. Brain Res. Rev. 2001, 37, 3–12. 10.1016/S0165-0173(01)00109-6. PubMed DOI

Mensah-Nyagan A. G.; Do-Rego J. L.; Beaujean D.; Luu-The V.; Pelletier G.; Vaudry H. Neurosteroids: expression of steroidogenic enzymes and regulation of steroid biosynthesis in the central nervous system. Pharmacol. Rev. 1999, 51, 63–81. PubMed

Jo D. H.; Abdallah M. A.; Young J.; Baulieu E. E.; Robel P. Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain. Steroids 1989, 54, 287–297. 10.1016/0039-128X(89)90003-2. PubMed DOI

Orchinik M.; Murray T. F.; Franklin P. H.; Moore F. L. Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 3830–3834. 10.1073/pnas.89.9.3830. PubMed DOI PMC

Ramirez V. D.; Zheng J. Membrane sex-steroid receptors in the brain. Front. Neuroendocrinol. 1996, 17, 402–439. 10.1006/frne.1996.0011. PubMed DOI

Klangkalya B.; Chan A. Structure-activity relationships of steroid hormones on muscarinic receptor binding. J. Steroid Biochem. 1988, 29, 111–118. 10.1016/0022-4731(88)90384-6. PubMed DOI

Majewska M. D. Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog. Neurobiol. 1992, 38, 379–394. 10.1016/0301-0082(92)90025-A. PubMed DOI

Valera S.; Ballivet M.; Bertrand D. Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 9949–9953. 10.1073/pnas.89.20.9949. PubMed DOI PMC

Wu F. S.; Gibbs T. T.; Farb D. H. Pregnenolone sulfate: a positive allosteric modulator at the N-methyl-D- aspartate receptor. Mol. Pharmacol. 1991, 40, 333–336. PubMed

Majewska M. D.; Schwartz R. D. Pregnenolone-sulfate: an endogenous antagonist of the gamma- aminobutyric acid receptor complex in brain?. Brain Res. 1987, 404, 355–360. 10.1016/0006-8993(87)91394-1. PubMed DOI

Adla S. K.; Slavikova B.; Smidkova M.; Tloustova E.; Svoboda M.; Vyklicky V.; Krausova B.; Hubalkova P.; Nekardova M.; Holubova K.; Vales K.; Budesinsky M.; Vyklicky L.; Chodounska H.; Kudova E. Physicochemical and biological properties of novel amide-based steroidal inhibitors of NMDA receptors. Steroids 2017, 117, 52–61. 10.1016/j.steroids.2016.08.010. PubMed DOI

Slavikova B.; Chodounska H.; Nekardova M.; Vyklicky V.; Ladislav M.; Hubalkova P.; Krausova B.; Vyklicky L.; Kudova E. Neurosteroid-like Inhibitors of N-Methyl-d-aspartate Receptor: Substituted 2-Sulfates and 2-Hemisuccinates of Perhydrophenanthrene. J. Med. Chem. 2016, 59, 4724.10.1021/acs.jmedchem.6b00079. PubMed DOI

Kudova E.; Chodounska H.; Slavikova B.; Budesinsky M.; Nekardova M.; Vyklicky V.; Krausova B.; Svehla P.; Vyklicky L. A New Class of Potent N-Methyl-D-Aspartate Receptor Inhibitors: Sulfated Neuroactive Steroids with Lipophilic D-Ring Modifications. J. Med. Chem. 2015, 58, 5950–5966. 10.1021/acs.jmedchem.5b00570. PubMed DOI

Cerny J.; Bozikova P.; Balik A.; Marques S. M.; Vyklicky L. NMDA Receptor Opening and Closing-Transitions of a Molecular Machine Revealed by Molecular Dynamics. Biomolecules 2019, 9, 546.10.3390/biom9100546. PubMed DOI PMC

Borovska J.; Vyklicky V.; Stastna E.; Kapras V.; Slavikova B.; Horak M.; Chodounska H.; Vyklicky L. Access of inhibitory neurosteroids to the NMDA receptor. Br. J. Pharmacol. 2012, 166, 1069–1083. 10.1111/j.1476-5381.2011.01816.x. PubMed DOI PMC

Stastna E.; Chodounska H.; Pouzar V.; Kapras V.; Borovska J.; Cais O.; Vyklicky L. Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA receptor responses in cultured rat hippocampal neurons. Steroids 2009, 74, 256–263. 10.1016/j.steroids.2008.11.011. PubMed DOI

Vyklicky V.; Smejkalova T.; Krausova B.; Balik A.; Korinek M.; Borovska J.; Horak M.; Chvojkova M.; Kleteckova L.; Vales K.; Cerny J.; Nekardova M.; Chodounska H.; Kudova E.; Vyklicky L. Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives. J. Neurosci. 2016, 36, 2161–2175. 10.1523/JNEUROSCI.3181-15.2016. PubMed DOI PMC

Rambousek L.; Bubenikova-Valesova V.; Kacer P.; Syslova K.; Kenney J.; Holubova K.; Najmanova V.; Zach P.; Svoboda J.; Stuchlik A.; Chodounska H.; Kapras V.; Adamusova E.; Borovska J.; Vyklicky L.; Vales K. Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-d-aspartate receptor 3alpha5beta-pregnanolone glutamate. Neuropharmacology 2011, 61, 61–68. 10.1016/j.neuropharm.2011.02.018. PubMed DOI

Vales K.; Rambousek L.; Holubova K.; Svoboda J.; Bubenikova-Valesova V.; Chodounska H.; Vyklicky L.; Stuchlik A. 3alpha5beta-Pregnanolone glutamate, a use-dependent NMDA antagonist, reversed spatial learning deficit in an animal model of schizophrenia. Behav. Brain Res. 2012, 235, 82–88. 10.1016/j.bbr.2012.07.020. PubMed DOI

Ishikawa M.; Yoshitomi T.; Covey D. F.; Zorumski C. F.; Izumi Y. Neurosteroids and oxysterols as potential therapeutic agents for glaucoma and Alzheimer’s disease. Neuropsychiatry 2018, 8, 344–359. 10.4172/Neuropsychiatry.1000356. PubMed DOI PMC

Guennoun R.; Labombarda F.; Gonzalez Deniselle M. C.; Liere P.; De Nicola A. F.; Schumacher M. Progesterone and allopregnanolone in the central nervous system: response to injury and implication for neuroprotection. J. Steroid Biochem. Mol. Biol. 2015, 146, 48–61. 10.1016/j.jsbmb.2014.09.001. PubMed DOI

Shafizadeh T. B.; Halsted C. H. gamma-Glutamyl hydrolase, not glutamate carboxypeptidase II, hydrolyzes dietary folate in rat small intestine. J. Nutr. 2007, 137, 1149–1153. 10.1093/jn/137.5.1149. PubMed DOI

Payne A. H.; Hales D. B. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr. Rev. 2004, 25, 947–970. 10.1210/er.2003-0030. PubMed DOI

Le Goascogne C.; Sananes N.; Gouezou M.; Takemori S.; Kominami S.; Baulieu E. E.; Robel P. Immunoreactive cytochrome P-450(17 alpha) in rat and guinea-pig gonads, adrenal glands and brain. J. Reprod. Fertil. 1991, 93, 609–622. 10.1530/jrf.0.0930609. PubMed DOI

Vianello S.; Waterman M. R.; Dalla Valle L.; Colombo L. Developmentally regulated expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 in rat liver. Endocrinology 1997, 138, 3166–3174. 10.1210/endo.138.8.5297. PubMed DOI

Dalla Valle L.; Toffolo V.; Vianello S.; Belvedere P.; Colombo L. Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span. J. Steroid Biochem. Mol. Biol. 2004, 91, 49–58. 10.1016/j.jsbmb.2004.01.008. PubMed DOI

Mukai H.; Tsurugizawa T.; Ogiue-Ikeda M.; Murakami G.; Hojo Y.; Ishii H.; Kimoto T.; Kawato S. Local neurosteroid production in the hippocampus: influence on synaptic plasticity of memory. Neuroendocrinology 2006, 84, 255–263. 10.1159/000097747. PubMed DOI

Missaghian E.; Kempna P.; Dick B.; Hirsch A.; Alikhani-Koupaei R.; Jegou B.; Mullis P. E.; Frey B. M.; Fluck C. E. Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents. J. Endocrinol. 2009, 202, 99–109. 10.1677/JOE-08-0353. PubMed DOI

Emanuelsson I.; Almokhtar M.; Wikvall K.; Gronbladh A.; Nylander E.; Svensson A. L.; Fex Svenningsen A.; Norlin M. Expression and regulation of CYP17A1 and 3beta-hydroxysteroid dehydrogenase in cells of the nervous system: Potential effects of vitamin D on brain steroidogenesis. Neurochem. Int. 2018, 113, 46–55. 10.1016/j.neuint.2017.11.007. PubMed DOI

Luu-The V. Assessment of steroidogenesis and steroidogenic enzyme functions. J. Steroid Biochem. Mol. Biol. 2013, 137, 176–182. 10.1016/j.jsbmb.2013.05.017. PubMed DOI

Miller W. L.; Auchus R. J. The ″backdoor pathway″ of androgen synthesis in human male sexual development. PLoS Biol. 2019, 17, e300019810.1371/journal.pbio.3000198. PubMed DOI PMC

UniProt C. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021, 49, D480–D489. 10.1093/nar/gkaa1100. PubMed DOI PMC

Kudova E.; Mares P.; Hill M.; Vondrakova K.; Tsenov G.; Chodounska H.; Kubova H.; Vales K. The neuroactive steroid pregnanolone glutamate: Anticonvulsant effect, metabolites and its effect on neurosteroid levels in developing rat brains. Pharmaceuticals 2022, 15, 49.10.3390/ph15010049. PubMed DOI PMC

Penning T. M.; Wangtrakuldee P.; Auchus R. J. Structural and Functional Biology of Aldo-Keto Reductase Steroid-Transforming Enzymes. Endocr. Rev. 2019, 40, 447–475. 10.1210/er.2018-00089. PubMed DOI PMC

Eechaute W. P.; Dhooge W. S.; Gao C. Q.; Calders P.; Rubens R.; Weyne J.; Kaufman J. M. Progesterone-transforming enzyme activity in the hypothalamus of the male rat. J. Steroid Biochem. Mol. Biol. 1999, 70, 159–167. 10.1016/S0960-0760(99)00106-5. PubMed DOI

Stell B. M.; Brickley S. G.; Tang C. Y.; Farrant M.; Mody I. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 14439–14444. 10.1073/pnas.2435457100. PubMed DOI PMC

Vicini S.; Losi G.; Homanics G. E. GABA(A) receptor delta subunit deletion prevents neurosteroid modulation of inhibitory synaptic currents in cerebellar neurons. Neuropharmacology 2002, 43, 646–650. 10.1016/S0028-3908(02)00126-0. PubMed DOI

Covey D. F.; Nathan D.; Kalkbrenner M.; Nilsson K. R.; Hu Y.; Zorumski C. F.; Evers A. S. Enantioselectivity of pregnanolone-induced gamma-aminobutyric acid(A) receptor modulation and anesthesia. J. Pharmacol. Exp. Ther. 2000, 293, 1009–1016. PubMed

Le Foll F.; Castel H.; Louiset E.; Vaudry H.; Cazin L. Multiple modulatory effects of the neuroactive steroid pregnanolone on GABAA receptor in frog pituitary melanotrophs. J. Physiol. 1997, 504, 387–400. 10.1111/j.1469-7793.1997.387be.x. PubMed DOI PMC

Lambert J. J.; Belelli D.; Hill-Venning C.; Peters J. A. Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci. 1995, 16, 295–303. 10.1016/S0165-6147(00)89058-6. PubMed DOI

Sooksawate T.; Simmonds M. A. Increased membrane cholesterol reduces the potentiation of GABA(A) currents by neurosteroids in dissociated hippocampal neurones. Neuropharmacology 1998, 37, 1103–1110. 10.1016/S0028-3908(98)00113-0. PubMed DOI

Li P.; Bracamontes J. R.; Manion B. D.; Mennerick S.; Steinbach J. H.; Evers A. S.; Akk G. The neurosteroid 5beta-pregnan-3alpha-ol-20-one enhances actions of etomidate as a positive allosteric modulator of alpha1beta2gamma2L GABAA receptors. Br. J. Pharmacol. 2014, 171, 5446–5457. 10.1111/bph.12861. PubMed DOI PMC

Park-Chung M.; Malayev A.; Purdy R. H.; Gibbs T. T.; Farb D. H. Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites. Brain Res. 1999, 830, 72–87. 10.1016/S0006-8993(99)01381-5. PubMed DOI

Wang M.; He Y.; Eisenman L. N.; Fields C.; Zeng C. M.; Mathews J.; Benz A.; Fu T.; Zorumski E.; Steinbach J. H.; Covey D. F.; Zorumski C. F.; Mennerick S. 3beta -hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists. J. Neurosci. 2002, 22, 3366–3375. 10.1523/JNEUROSCI.22-09-03366.2002. PubMed DOI PMC

Callachan H.; Cottrell G. A.; Hather N. Y.; Lambert J. J.; Nooney J. M.; Peters J. A. Modulation of the GABAA receptor by progesterone metabolites. Proc. R. Soc. London, Ser. B 1987, 231, 359–369. 10.1098/rspb.1987.0049. PubMed DOI

Collins G. G. Effects of the anaesthetic 2,6-diisopropylphenol on synaptic transmission in the rat olfactory cortex slice. Br. J. Pharmacol. 1988, 95, 939–949. 10.1111/j.1476-5381.1988.tb11724.x. PubMed DOI PMC

Vicini S.; Mienville J. M.; Costa E. Actions of benzodiazepine and beta-carboline derivatives on gamma-aminobutyric acid-activated Cl- channels recorded from membrane patches of neonatal rat cortical neurons in culture. J. Pharmacol. Exp. Ther. 1987, 243, 1195–1201. PubMed

Germann A. L.; Pierce S. R.; Burbridge A. B.; Steinbach J. H.; Akk G. Steady-State Activation and Modulation of the Concatemeric alpha1beta2gamma2L GABA(A) Receptor. Mol. Pharmacol. 2019, 96, 320–329. 10.1124/mol.119.116913. PubMed DOI PMC

Walsh R. N.; Cummins R. A. The Open-Field Test: a critical review. Psychol. Bull. 1976, 83, 482–504. 10.1037/0033-2909.83.3.482. PubMed DOI

Kallai J.; Makany T.; Csatho A.; Karadi K.; Horvath D.; Kovacs-Labadi B.; Jarai R.; Nadel L.; Jacobs J. W. Cognitive and affective aspects of thigmotaxis strategy in humans. Behav. Neurosci. 2007, 121, 21–30. 10.1037/0735-7044.121.1.21. PubMed DOI

Treit D.; Fundytus M. Thigmotaxis as a test for anxiolytic activity in rats. Pharmacol. Biochem. Behav. 1988, 31, 959–962. 10.1016/0091-3057(88)90413-3. PubMed DOI

Schnorr S. J.; Steenbergen P. J.; Richardson M. K.; Champagne D. L. Measuring thigmotaxis in larval zebrafish. Behav. Brain Res. 2012, 228, 367–374. 10.1016/j.bbr.2011.12.016. PubMed DOI

MacPhail R. C.; Brooks J.; Hunter D. L.; Padnos B.; Irons T. D.; Padilla S. Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 2009, 30, 52–58. 10.1016/j.neuro.2008.09.011. PubMed DOI

Cahill G. M.; Hurd M. W.; Batchelor M. M. Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 1998, 9, 3445–3449. 10.1097/00001756-199810260-00020. PubMed DOI

Akk G.; Steinbach J. H. Activation and block of recombinant GABA(A) receptors by pentobarbitone: a single-channel study. Br. J. Pharmacol. 2000, 130, 249–258. 10.1038/sj.bjp.0703335. PubMed DOI PMC

MacDonald J. F.; Miljkovic Z.; Pennefather P. Use-dependent block of excitatory amino acid currents in cultured neurons by ketamine. J. Neurophysiol. 1987, 58, 251–266. 10.1152/jn.1987.58.2.251. PubMed DOI

Sze Y.; Gill A. C.; Brunton P. J. Sex-dependent changes in neuroactive steroid concentrations in the rat brain following acute swim stress. J. Neuroendocrinol. 2018, 30, e1264410.1111/jne.12644. PubMed DOI PMC

Kancheva R.; Hill M.; Novak Z.; Chrastina J.; Kancheva L.; Starka L. Neuroactive steroids in periphery and cerebrospinal fluid. Neuroscience 2011, 191, 22–27. 10.1016/j.neuroscience.2011.05.054. PubMed DOI

Irwin R. W.; Solinsky C. M.; Loya C. M.; Salituro F. G.; Rodgers K. E.; Bauer G.; Rogawski M. A.; Brinton R. D. Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer’s disease. PLoS One 2015, 10, e012831310.1371/journal.pone.0128313. PubMed DOI PMC

Bixo M.; Andersson A.; Winblad B.; Purdy R. H.; Backstrom T. Progesterone, 5alpha-pregnane-3,20-dione and 3alpha-hydroxy-5alpha-pregnane-20-one in specific regions of the human female brain in different endocrine states. Brain Res. 1997, 764, 173–178. 10.1016/S0006-8993(97)00455-1. PubMed DOI

Liere P.; Cornil C. A.; de Bournonville M. P.; Pianos A.; Keller M.; Schumacher M.; Balthazart J. Steroid profiles in quail brain and serum: Sex and regional differences and effects of castration with steroid replacement. J. Neuroendocrinol. 2019, 31, e1268110.1111/jne.12681. PubMed DOI PMC

Qaiser M. Z.; Dolman D. E. M.; Begley D. J.; Abbott N. J.; Cazacu-Davidescu M.; Corol D. I.; Fry J. P. Uptake and metabolism of sulphated steroids by the blood-brain barrier in the adult male rat. J. Neurochem. 2017, 142, 672–685. 10.1111/jnc.14117. PubMed DOI PMC

Represa A.; Ben-Ari Y. Trophic actions of GABA on neuronal development. Trends Neurosci. 2005, 28, 278–283. 10.1016/j.tins.2005.03.010. PubMed DOI

Grobin A. C.; Gizerian S.; Lieberman J. A.; Morrow A. L. Perinatal allopregnanolone influences prefrontal cortex structure, connectivity and behavior in adult rats. Neuroscience 2006, 138, 809–819. 10.1016/j.neuroscience.2005.12.026. PubMed DOI

Zorumski C. F.; Paul S. M.; Covey D. F.; Mennerick S. Neurosteroids as novel antidepressants and anxiolytics: GABA-A receptors and beyond. Neurobiol. Stress 2019, 11, 10019610.1016/j.ynstr.2019.100196. PubMed DOI PMC

Reddy D. S.; Estes W. A. Clinical Potential of Neurosteroids for CNS Disorders. Trends Pharmacol. Sci. 2016, 37, 543–561. 10.1016/j.tips.2016.04.003. PubMed DOI PMC

Kharasch E. D.; Hollmann M. W. Steroid Anesthesia Revisited: Again. Anesth. Analg. 2015, 120, 983–984. 10.1213/ANE.0000000000000667. PubMed DOI PMC

Smejkalova T.; Korinek M.; Krusek J.; Hrcka Krausova B.; Candelas Serra M.; Hajdukovic D.; Kudova E.; Chodounska H.; Vyklicky L. Endogenous neurosteroids pregnanolone and pregnanolone sulfate potentiate presynaptic glutamate release through distinct mechanisms. Br. J. Pharmacol. 2021, 178, 3888–3904. 10.1111/bph.15529. PubMed DOI PMC

Laverty D.; Thomas P.; Field M.; Andersen O. J.; Gold M. G.; Biggin P. C.; Gielen M.; Smart T. G. Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites. Nat. Struct. Mol. Biol. 2017, 24, 977–985. 10.1038/nsmb.3477. PubMed DOI PMC

Weir C. J.; Ling A. T.; Belelli D.; Wildsmith J. A.; Peters J. A.; Lambert J. J. The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors. Br. J. Anaesth. 2004, 92, 704–711. 10.1093/bja/aeh125. PubMed DOI

Kysilov B.; Hrcka Krausova B.; Vyklicky V.; Smejkalova T.; Korinek M.; Horak M.; Chodounska H.; Kudova E.; Cerny J.; Vyklicky L. Pregnane-based steroids are novel positive NMDA receptor modulators that may compensate for the effect of loss-of-function disease-associated GRIN mutations. Br. J. Pharmacol. 2022, 179, 3970–3990. 10.1111/bph.15841. PubMed DOI

Selye H. Anesthetic Effect of Steroid Hormones. Proc. Soc. Exp. Biol. Med. 1941, 46, 116–121. 10.3181/00379727-46-11907. DOI

MacDonald J. F.; Bartlett M. C.; Mody I.; Pahapill P.; Reynolds J. N.; Salter M. W.; Schneiderman J. H.; Pennefather P. S. Actions of ketamine, phencyclidine and MK-801 on NMDA receptor currents in cultured mouse hippocampal neurones. J. Physiol. 1991, 432, 483–508. 10.1113/jphysiol.1991.sp018396. PubMed DOI PMC

Green S. M.; Roback M. G.; Kennedy R. M.; Krauss B. Clinical practice guideline for emergency department ketamine dissociative sedation: 2011 update. Ann. Emerg. Med. 2011, 57, 449–461. 10.1016/j.annemergmed.2010.11.030. PubMed DOI

Zanos P.; Moaddel R.; Morris P. J.; Riggs L. M.; Highland J. N.; Georgiou P.; Pereira E. F. R.; Albuquerque E. X.; Thomas C. J.; Zarate C. A. Jr.; Gould T. D. Ketamine and Ketamine Metabolite Pharmacology: Insights into Therapeutic Mechanisms. Pharmacol. Rev. 2018, 70, 621–660. 10.1124/pr.117.015198. PubMed DOI PMC

Morris P. J.; Moaddel R.; Zanos P.; Moore C. E.; Gould T. D.; Zarate C. A.; Thomas C. J. Jr.; Thomas C. J. Synthesis and N-Methyl-d-aspartate (NMDA) Receptor Activity of Ketamine Metabolites. Org. Lett. 2017, 19, 4572–4575. 10.1021/acs.orglett.7b02177. PubMed DOI PMC

Chen J.; Patel R.; Friedman T. C.; Jones K. S. The Behavioral and Pharmacological Actions of NMDA Receptor Antagonism are Conserved in Zebrafish Larvae. Int. J. Comp. Psychol. 2010, 23, 82–90. PubMed PMC

Li F.; Lin J.; Liu X.; Li W.; Ding Y.; Zhang Y.; Zhou S.; Guo N.; Li Q. Characterization of the locomotor activities of zebrafish larvae under the influence of various neuroactive drugs. Ann. Transl. Med. 2018, 6, 173.10.21037/atm.2018.04.25. PubMed DOI PMC

Zoodsma J. D.; Chan K.; Bhandiwad A. A.; Golann D. R.; Liu G.; Syed S. A.; Napoli A. J.; Burgess H. A.; Sirotkin H. I.; Wollmuth L. P. A Model to Study NMDA Receptors in Early Nervous System Development. J. Neurosci. 2020, 40, 3631–3645. 10.1523/JNEUROSCI.3025-19.2020. PubMed DOI PMC

Hill M.; Hana V. Jr; Velikova M.; Parizek A.; Kolatorova L.; Vitku J.; Skodova T.; Simkova M.; Simjak P.; Kancheva R.; Koucky M.; Kokrdova Z.; Adamcova K.; Cerny A.; Hajek Z.; Duskova M.; Bulant J.; Starka L. A method for determination of one hundred endogenous steroids in human serum by gas chromatography-tandem mass spectrometry. Physiol. Res. 2019, 68, 179–207. 10.33549/physiolres.934124. PubMed DOI

Vyklicky V.; Korinek M.; Balik A.; Smejkalova T.; Krausova B.; Vyklicky L.. Analysis of Whole-Cell NMDA Receptor Currents. In Ionotropic Glutamate Receptor Technologies; Springer, 2016; pp 205–219.

Westerfield M.The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th ed.; University of Oregon Press: Eugene, 2000.

Alestrom P.; D’Angelo L.; Midtlyng P. J.; Schorderet D. F.; Schulte-Merker S.; Sohm F.; Warner S. Zebrafish: Housing and husbandry recommendations. Lab. Anim. 2020, 54, 213–224. 10.1177/0023677219869037. PubMed DOI PMC

Effect of Treatment on Steroidome in Women with Multiple Sclerosis