Cao LQ, Montana MC, Germann AL, Shin DJ, Chakrabarti S, Mennerick S, Yuede CM, Wozniak DF, Evers AS, Akk G.

R01 GM108580 NIGMS NIH HHS , R21 MH111461 NIMH NIH HHS, R21 MH104506 NIMH NIH HHS, R01 GM108799 NIGMS NIH HHS , T32 GM108539 NIGMS NIH HHS

	Figure 1. Effects of combining alfaxalone with propofol, or with diazepam on mouse behavior. (A) A 30-min activity test was conducted immediately following i.p. injections of normal saline (20 Units), propofol (50 mg/kg), alfaxalone (30 mg/kg), or the combination of propofol (50 mg/kg) + alfaxalone (30 mg/kg), to determine drug effects on time at rest (absence of movement) as a function of post-injection time (5-min blocks) across the test session (means ± S.E.M.; n = 11–12 mice per treatment group). A repeated measures (rm) ANOVA revealed a significant drug effect (**p = 0.0005) and drug × time interaction (***p < 0.00005), with the propofol + alfaxalone mice exhibiting significantly increased time at rest compared to the single drug/saline control groups on average across the test session. Significant between-groups comparisons are shown for these contrasts: saline vs. propofol + alfaxalone at 5, 10, 15 (*p < 0.003), and 20 (*p = 0.031) min post-injection; alfaxalone vs. propofol + alfaxalone at 5, 10 (†p < 0.002), and 20 (†p = 0.038) min; and propofol vs. propofol + alfaxalone at 5, 10 (#p < 0.0002), and 15 and 20 (#p < 0.040) min. No significant differences were observed for the contrasts involving the saline vs. propofol or saline vs. alfaxalone groups. (B) A separate study was conducted on an independent cohort of naïve mice to examine the potential sedating drug effects following i.p. injections of normal saline (20 Units), diazepam (4 mg/kg), alfaxalone (30 mg/kg), or the combination of diazepam (4 mg/kg) + alfaxalone (30 mg/kg) on time at rest (n = 10 for each group). An rmANOVA yielded a significant drug effect (**p < 0.00005) and drug × time interaction (***p = 0.0004), with the diazepam + alfaxalone treated mice having significantly greater times at rest compared to the saline, alfaxalone, and diazepam groups on average across the session. Between-groups comparisons conducted within these contrasts showed robust differences between each of the single drug/saline control groups relative to the diazepam + alfaxalone treated mice for every post-injection time (5–30 min) interval (p < 0.0002; for the comparisons involving saline (*), alfaxalone (†), or diazepam (#), respectively). The diazepam group also had significantly increased rest times relative to the saline control mice, with pair-wise comparisons revealing significant differences at 10, 15, 20, and 25-min post-injection (††p < 0.003).
	Figure 2. Comparison of the behavioral effects of from the co-administration of drugs with the sum of effects of individual drugs. (A) The graph compares the performance levels induced by the co-administration of propofol + alfaxalone to that resulting from the mathematical sums of the single drug applications. The magnitude of change in the mean percent times at rest above baseline for the co-administration of the drugs was greater early on in the test session, being approximately 7 times higher during the first time interval compared to that derived from adding together the percentages of the single applications of propofol and alfaxalone. These differences declined over the session such that the percentages from the two conditions were roughly equivalent by the end of the test session. (B) The graph compares the performance levels induced by the co-administration of diazepam + alfaxalone to that resulting from the mathematical sums of the single drug applications. The percent time at rest above baseline was consistently higher throughout the test session in the co-administered group compared to the percent change observed in the summed, single drug applications. The degree of change in the percent times at rest above baseline ranged from being 8.3 to 1.4 times higher in the co-administered drug group.
	Figure 3. Effects of alfaxalone, propofol and diazepam on synaptic currents. Sample averaged sIPSCs recorded under control conditions, or in the presence of 10 nM alfaxalone (ALF), 300 nM alfaxalone, 10 nM propofol (PRO), 1 μM propofol, 3 nM diazepam (DZP), 1 μM diazepam, or the combinations of propofol + alfaxalone or diazepam + alfaxalone. The drugs were added to the bath at least 10 min before recordings. All traces are from separate cells.
	Figure 4. Summary of the effects of alfaxalone, propofol and diazepam on synaptic currents. (A) Summary of the decay time courses of sIPSCs recorded under control conditions or in the presence of 10 nM or 300 nM alfaxalone. The graph shows data from each cell tested (open circles) and mean ± S.D. for the experimental condition (filled circles and error bars). (B) Summary of the decay time courses of sIPSCs recorded in the presence of propofol with or without alfaxalone. The open symbols show data from each cell tested, and the filled symbols and error bars show mean ± S.D. Due to saturation at lower propofol concentrations, 3 μM propofol was not tested in the presence of 300 nM alfaxalone. Statistical analysis was done by comparing the decay times for 10 nM-3 μM propofol to control, using ANOVA with Dunnett’s correction. For combinations of propofol and alfaxalone, the top symbol applies to comparison of propofol + alfaxalone with alfaxalone alone. The bottom symbol applies to comparison of propofol + alfaxalone with propofol alone. (C) Summary of the decay time courses of sIPSCs recorded in the presence of diazepam with or without alfaxalone. The open symbols show data from each cell tested, and the filled symbols and error bars show mean ± S.D. Statistical analysis was done by comparing the decay times for 1 nM-1 μM diazepam to control. For combinations of diazepam and alfaxalone and, the top symbol applies to comparison of diazepam + alfaxalone with alfaxalone alone. The bottom symbol applies to comparison of diazepam + alfaxalone with diazepam alone. In (B) and (C), the solid line shows the decay time under control conditions (no modulators). The dashed line shows the decay time in the presence of 10 nM alfaxalone. The dotted line shows the decay time in the presence of 300 nM alfaxalone. The number of cells was 3–17 for control, drug, or drug combinations. #not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
	Figure 5. Activation and modulation of concatemeric α1β2γ2L GABAA receptors. (A) Direct activation of the receptor by GABA, propofol (PRO), or alfaxalone (ALF). The ordinate shows the estimated open probability (Po). The curves for GABA and propofol were generated by fitting Equation (1) to experimental data. Direct activation by alfaxalone produced very small currents (<100 nA at 30 μM). The predicted activation curve for alfaxalone was generated using KALF and cALF values determined in (B). (B) Potentiation of GABA-activated receptors by propofol, alfaxalone, or diazepam (DZP). The solid curves were generated by fitting Equation (1) to the data. L was held at (1 − Po,2 μM GABA)/Po,2 μM GABA. The dashed line shows the predicted propofol-potentiation curve using KPRO and cPRO values from (A). (C) Activation by GABA in the presence of 1 μM diazepam, 1 μM alfaxalone or the combination of diazepam + alfaxalone. The curves for single modulators were generated by fitting Equation (1) to the data. L was held at at (1 − Po,1 μM ALF)/Po,1 μM ALF or at (1 − Po,1 μM DZP)/Po,1 μM DZP . The open probability for receptors activated by 1 μM alfaxalone or 1 μM diazepam was calculated from the KALF and cALF, or KDZP and cDZP values estimated in (B). The simulated curve for the combination of diazepam + alfaxalone was calculated using Equation (1) and the KGABA and cGABA values from (A), and KALF, cALF, KDZP and cDZP values from (B). (D) Activation by GABA in the presence of 10 μM propofol, 1 μM alfaxalone or the combination of propofol + alfaxalone. The curve for propofol (green dashed line) is based on data from a previous report45. The data for alfaxalone are reproduced from panel C. The simulated curve for the combination of propofol + alfaxalone was calculated using Equation (1) and the KGABA, cGABA, KPRO and cPRO values from (A), and KALF and cALF values from (B). In (C) and (D), the black dotted lines show the GABA concentration-response relationship in the absence of modulators (from A). In all panels the data points show mean ± S.D. from at least five cells.

Akk, GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC50 and Basal Activity. 2018, Pubmed, Xenbase

Akk, GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC50 and Basal Activity. 2018, Pubmed , Xenbase
Amin, GABAA receptor needs two homologous domains of the beta-subunit for activation by GABA but not by pentobarbital. 1993, Pubmed , Xenbase
Baumann, Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement. 2002, Pubmed , Xenbase
Bracamontes, Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site. 2011, Pubmed , Xenbase
Caudle, The misuse of analysis of variance to detect synergy in combination drug studies. 1993, Pubmed
Chakrabarti, Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABA(A) Receptors. 2016, Pubmed
Chang, Allosteric activation mechanism of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine. 1999, Pubmed , Xenbase
Claeys, Haemodynamic changes during anaesthesia induced and maintained with propofol. 1988, Pubmed
Drexler, Synergistic Modulation of γ-Aminobutyric Acid Type A Receptor-Mediated Synaptic Inhibition in Cortical Networks by Allopregnanolone and Propofol. 2016, Pubmed
Dundee, Anterograde amnesic effects of pethidine, hyoscine and diazepam in adults. 1972, Pubmed
Eaton, Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol. 2016, Pubmed
Emnett, A Clickable Analogue of Ketamine Retains NMDA Receptor Activity, Psychoactivity, and Accumulates in Neurons. 2016, Pubmed
Emnett, Interaction between positive allosteric modulators and trapping blockers of the NMDA receptor channel. 2015, Pubmed
Essrich, Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. 1998, Pubmed
Fischer, Anticonflict and reinforcing effects of triazolam + pregnanolone combinations in rhesus monkeys. 2011, Pubmed
Forman, Monod-Wyman-Changeux allosteric mechanisms of action and the pharmacology of etomidate. 2012, Pubmed
Forman, Mutations in the GABAA receptor that mimic the allosteric ligand etomidate. 2012, Pubmed , Xenbase
Franks, Molecular targets underlying general anaesthesia. 2006, Pubmed
Gasior, Anticonvulsant and behavioral effects of neuroactive steroids alone and in conjunction with diazepam. 1997, Pubmed
Gunter, Benzodiazepine and neuroactive steroid combinations in rats: anxiolytic-like and discriminative stimulus effects. 2016, Pubmed
Günther, Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors. 1995, Pubmed
Hernandez, GABA A Receptor Coupling Junction and Pore GABRB3 Mutations are Linked to Early-Onset Epileptic Encephalopathy. 2017, Pubmed
Horne, The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation. 1993, Pubmed
Hosie, Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. 2006, Pubmed
Jurd, General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. 2003, Pubmed
Lam, Modulatory and direct effects of propofol on recombinant GABAA receptors expressed in xenopus oocytes: influence of alpha- and gamma2-subunits. 1998, Pubmed , Xenbase
Li, The neurosteroid 5β-pregnan-3α-ol-20-one enhances actions of etomidate as a positive allosteric modulator of α1β2γ2L GABAA receptors. 2014, Pubmed , Xenbase
Li, Natural and enantiomeric etiocholanolone interact with distinct sites on the rat alpha1beta2gamma2L GABAA receptor. 2007, Pubmed
Loeffler, Oral benzodiazepines and conscious sedation: a review. 1992, Pubmed
Macdonald, GABAA receptor channels. 1994, Pubmed
Mangan, Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAA Receptors. 2005, Pubmed
Mennerick, Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures. 1995, Pubmed
Mihic, A single amino acid of the human gamma-aminobutyric acid type A receptor gamma 2 subunit determines benzodiazepine efficacy. 1994, Pubmed , Xenbase
Mitchell, GABAA and glycine receptor-mediated transmission in rat lamina II neurones: relevance to the analgesic actions of neuroactive steroids. 2007, Pubmed
MONOD, ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. 1965, Pubmed
Olsen, GABA A receptors: subtypes provide diversity of function and pharmacology. 2009, Pubmed
Pritchett, Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. 1989, Pubmed
Puia, Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gamma-aminobutyric acid-gated Cl- currents. 1991, Pubmed
Reynolds, Sedation and anesthesia mediated by distinct GABA(A) receptor isoforms. 2003, Pubmed
Richards, Additive and non-additive effects of mixtures of short-acting intravenous anaesthetic agents and their significance for theories of anaesthesia. 1981, Pubmed
Ruesch, An allosteric coagonist model for propofol effects on α1β2γ2L γ-aminobutyric acid type A receptors. 2012, Pubmed , Xenbase
Sanna, Actions of the general anesthetic propofol on recombinant human GABAA receptors: influence of receptor subunits. 1995, Pubmed , Xenbase
Shin, Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABAA Receptors. 2018, Pubmed , Xenbase
Shin, The Actions of Drug Combinations on the GABAA Receptor Manifest as Curvilinear Isoboles of Additivity. 2017, Pubmed , Xenbase
Sigel, The benzodiazepine binding site of GABAA receptors. 1997, Pubmed
Skues, The pharmacology of propofol. 1989, Pubmed
Slinker, The statistics of synergism. 1998, Pubmed
Steinbach, Modulation of GABA(A) receptor channel gating by pentobarbital. 2001, Pubmed
Swanwick, Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons. 2006, Pubmed
Wozniak, Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults. 2004, Pubmed
Zimmerman, Potentiation of gamma-aminobutyric acidA receptor Cl- current correlates with in vivo anesthetic potency. 1994, Pubmed