PMC

Potentiation of miniature inhibitory postsynaptic currents by alphaxalone but not by 3β-OH. (a) Representative traces from CeM neurones before and after perfusion with 3β-OH (top panel) or alphaxalone (bottom panel). (b) Decay tau was increased only after perfusion of alphaxalone but not 3β-OH; alphaxalone increased decay tau from 26.7 (3.4) to 62.9 (6.6) ms (two-tailed paired t-test: t₍₁₀₎=6.82, P<0.001). (c) Left panel shows that cumulative frequency distribution for decay tau confirms no change after application of 3β-OH (control 1479 events, 3β-OH 1497 events); right panel shows longer decay taus after alphaxalone perfusion (1523 events for alphaxalone, 2134 events for the control). (d) Representative traces showing tonic GABA current after 3 μM 3β-OH (purple trace) or 3 μM alphaxalone (green trace) perfusion. (b) Difference from baseline showed a minimal effect of 3β-OH on tonic current in comparison with alphaxalone (3β-OH five cells, alphaxalone six cells; unpaired two-tailed t-test: t₍₉₎=2.31, P=0.046). ∗P<0.05, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; CeM, central medial nucleus; GABA, γ-aminobutyric acid A.

Alphaxalone interactions with nearby residues in molecular dynamics (MD) simulations. a A representative snapshot from MD simulations shows alphaxalone contacts with T306, Q242, and W246. Distances between alphaxalone and the residues are measured as marked by the dash lines. b Histograms of distances between alphaxalone and W246 atoms (1 and 2) shown in a. c Histograms of distances between alphaxalone and Q242 or T306 atoms (3 and 4, respectively) shown in a. Data in b and c are from three replicate 50-ns simulations, where snapshots were collected every 100-ps for analysis. Distances were measured for each of the five alphaxalone molecules per replicate simulation (500 snapshots × 3 replicates × 5 alphaxalone = 7500 distances in total)

Characterisation of hypnotic properties of alphaxalone, CDNC24, and propofol in postnatal Day 7 rat pups. (a) The chemical structure of tested agents: i) alphaxalone (5α-pregnan-3α-ol-11,20-dione), ii) CDNC24 ([3α,5α]-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-24-ol) and iii) propofol (2,6-diisopropylphenol). (b) Percentage of rat pups with loss of righting reflex (LORR) with increasing doses of alphaxalone (black), CDNC24 (blue), or propofol (red) after a single i. p. injection. The dose that caused LORR in 50% of animals (marked with horizontal dashed line in the graph) was used to determine the ED₅₀ (mg kg⁻¹) of alphaxalone, CDNC24, or propofol (exact ED₅₀ values for each of three agents are indicated above the graph in corresponding colours) (n=4–11 rats per each data point). (c) Duration of LORR (min) with increasing doses of alphaxalone (black), CDNC24 (blue), or propofol (red) after a single i. p. injection. The difference in the duration of LORR at the dose of 60 mg kg⁻¹ for agents tested is presented as a vertical dashed line in the graph. Each data point is presented as mean (standard error of the mean) (n=4–16 rats per each data point). The inset highlights the assessment of the equipotent doses using the duration of LORR (min) after a single i. p. injection. Alphaxalone (black) and CDNC24 (blue), 10 mg kg⁻¹ for both agents, are comparable with propofol (red) at 20 mg kg⁻¹, a dose known to cause significant developmental neuroapoptosis. The dashed line in the inserted graph indicates that chosen doses of tested agents caused LORR for ∼40 min. (d) The mortality rate of rat pups with increasing doses of alphaxalone (black), CDNC24 (blue), or propofol (red) after a single i. p. injection. The dose that caused mortality in 50% of animals (outlined as a horizontal dashed line in the graph) was used to determine LD₅₀ (mg kg⁻¹) of alphaxalone or propofol (exact ED₅₀ values for each of two agents are indicated above the graph in corresponding colours). Note that CDNC24 did not cause any mortality even at the highest dose tested (ED₅₀ >60 mg kg⁻¹). The difference in mortality rate at a dose of 60 mg kg⁻¹ for agents tested is indicated as a vertical dashed line in the graph (n=4–10 rats per each data point). (e) Therapeutic indices of alphaxalone, CDNC24, and propofol. The therapeutic indices of tested compounds were calculated as the ratio of corresponding LD₅₀ and ED₅₀ values.

a. Total distance traveled by WT and α4 KO mice in response to a low dose of alphaxalone (15 mg/kg) over a 10 min period. Genotypes did not differ in response to vehicle injection. Alphaxalone stimulated locomotor activity compared to vehicle in WT (p < 0.05) but not KO mice. b. Percent distance covered in the center zone of the open field by WT and α4 KO mice. No differences were observed between vehicle-treated and alphaxalone-treated WT and α4 KO animals. c. Total distance traveled in response to a low dose of etomidate (3 mg/kg). No differences were observed between WT and KO mice. Data are expressed as mean ± SEM.