A neurosteroid analogue with T-type calcium channel blocking properties is an effective hypnotic, but is not harmful to neonatal rat brain

Abstract

Background: More than 4 million children are exposed annually to sedatives and general anaesthetics (GAs) in the USA alone. Recent data suggest that common GAs can be detrimental to brain development causing neurodegeneration and long-term cognitive impairments. Challenged by a recent US Food and Drug Administration (FDA) warning about potentially neurotoxic effects of GAs in children, there is an urgent need to develop safer GAs.

Methods: Postnatal Day 7 (P7) rat pups of both sexes were exposed to six (repeated every 2 h) injections of equipotent hypnotic doses of ketamine or the neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) for 12 h. Loss of righting reflex was used to assess hypnotic properties and therapeutic index; quantitative caspase-3 immunohistochemistry was used to assess developmental neuroapoptosis; patch-clamp recordings in acute brain slices were used to assess the effects of 3β-OH on neuronal excitability and synaptic transmission. Cognitive abilities of rats exposed to ketamine, 3β-OH, or vehicle at P7 were assessed in young adulthood using the radial arm maze.

Results: The neuroactive steroid 3β-OH has a therapeutic index similar to ketamine, a commonly used clinical GA. We report that 3β-OH is safe and, unlike ketamine, does not cause neuroapoptosis or impair cognitive development when administered to P7 rat pups. Interestingly, 3β-OH blocks T-type calcium channels and presynaptically dampens synaptic transmission at hypnotically-relevant brain concentrations, but it lacks a direct effect on γ-aminobutyric acid A or glutamate-gated ion channels.

Conclusions: The neurosteroid 3β-OH is a relatively safe hypnotic that warrants further consideration for paediatric anaesthesia.

Keywords: calcium channels; developmental neurotoxicity; neurosteroid.

Fig 1

(A) (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), like ketamine, causes dose-dependent loss of righting reflex (LORR). Inset: 3β-OH is a neuroactive steroid analogue based on a progesterone ring where hydrogen bonding groups in positions 3 and 17 were substituted with OH and CN groups, respectively. Rats in each group received only one dose of either ketamine or 3β-OH (as shown on axis x). Vehicles, 15% β-cyclodextrin, or saline, did not cause LORR (n=8 per group). 3β-OH, ketamine, and ketamine+β-cyclodextrin groups induced a dose-dependent shortening of the time to LORR. 3β-OH had slower onset of LORR at each dose. Note that at 1 mg kg⁻¹, none of ketamine (KET), ketamine+β-cyclodextrin (KET+CYCLO) or 3β-OH produced LORR (n=6–26 pups per data point). (B) The percent of rat pups with LORR was used to calculate the ED₅₀ for LORR: 3.2 (0.1) mg kg⁻¹ with 3β-OH, 3.5 (0.2) mg kg⁻¹ with ketamine, and 4.1 (0.4) mg kg⁻¹ with ketamine+β-cyclodextrin (n=6–26 pups per data point). (C) The duration of LORR with 3β-OH, ketamine, or ketamine+β-cyclodextrin was dose-dependent, reaching more than 400 min with the highest doses. Calculated ED₅₀ was 39 (4) mg kg⁻¹ for 3β-OH, 67 (2) mg kg⁻¹ for ketamine, and 63 (2) mg kg⁻¹ for ketamine+β-cyclodextrin, suggesting an almost two-fold higher potency for 3β-OH. (D) The connected circles indicate that the estimated equipotent single dose of 3β-OH, which is comparable with a lower dose of ketamine (20 mg kg⁻¹, i.p.), was 5 mg kg⁻¹, i.p., whereas the estimated equipotent dose of 3β-OH comparable with a higher dose of ketamine (40 mg kg⁻¹, i.p.) was 10 mg kg⁻¹, i.p. The inset shows the mortality data in rat pups treated with 3β-OH or ketamine.

Fig 2

(3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), unlike ketamine, does not cause developmental neuroapoptosis at higher doses. 3β-OH (10 mg kg⁻¹, i.p.), ketamine (40 mg kg⁻¹, i.p.) and ketamine (40 mg kg⁻¹)+β-cyclodextrin were injected in P7 rat pups every 2 h for a total of six injections. Bar graphs on the left show averaged data from multiple experiments; representative images are depicted on the right panels. (A) The CA1-subiculum region exhibits a significant increase in caspase-3 activation when compared with groups treated with 3β-OH (3β-OH 10), ketamine alone (KET 40) or ketamine+β-cyclodextrin (KET 40+CYCLO) (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) suggesting that 3β-OH, unlike ketamine, does not cause significant developmental neuroapoptosis. This was confirmed by the finding that activated caspase-3 staining in 3β—OH—treated animals was not significantly upregulated compared with saline (SAL) or β-cyclodextrin (CYCLO) controls (P=0.964 vs saline and P=0.980 vs β-cyclodextrin) (n=6 pups per data point). (B) In thalamic anteroventral nucleus (TAV) there was minimal caspase-3 activation in vehicle groups, whilst there was a significant increase in caspase-3 activation in ketamine or ketamine+β-cyclodextrin groups compared with vehicle controls (***P<0.001). The level of caspase-3 activation in 3β-OH animals was comparable with vehicle controls and significantly lower than in either ketamine or ketamine+β-cyclodextrin (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) groups (n=6 pups per data point). (C) In thalamic lateral nucleus (TL) there was minimal caspase-3 activation in vehicle groups, whilst there was significant caspase-3 activation in ketamine or ketamine+β-cyclodextrin groups (**P=0.006 vs SAL and P=0.009 vs CYCLO). Note that the caspase-3 activation in the 3β-OH group is not significant compared with vehicle controls (n=6 pups per data point). (D) In the cingulate cortex, there was no difference in caspase-3 activation in the 3β-OH group compared with vehicle controls (P>0.999 vs SAL; P=0.995 vs CYCLO), but ketamine or ketamine+β-cyclodextrin groups exhibited significant increases in caspase-3 activation compared with vehicle control (***P<0.001 vs SAL and P<0.001 vs CYCLO). 3β-OH does not exhibit neurotoxic potential compared with either ketamine or ketamine+β-cyclodextrin groups (***P<0.001 vs KET 40 and P<0.001 vs KET 40+CYCLO) (n=6 pups per data point). All statistical analyses were done using one-way analysis of variance with Tukey's post hoc test. Representative photomicrographs shown in the right-side panels depict activated caspase-3 staining in ketamine+β-cyclodextrin compared with 3β-OH or a vehicle. Scale bars in the low magnification images in panels (A–C) are 400 μm, and 800 μm in panel (D). Scale bars in all high magnification insets are 100 μm.

Fig 3

(3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), unlike ketamine, does not impair spatial learning and memory. P7 rat pups were exposed to saline, β-cyclodextrin, or higher doses of ketamine or 3β-OH at P7 and tested for their spatial working memory using the eight-arm radial arm maze test (RAM) in young adulthood. (A) The overall appearance and daily weight of rats in each group (saline, β-cyclodextrin at 15%, 3β-OH at 10 mg kg⁻¹ or ketamine at 40 mg kg⁻¹, administered every 2 h for a total of six doses) were examined. Daily weight did not differ between groups. The time points when animals were treated (P7), food restricted (from P45), tested by RAM (from P53), and permitted to eat ad libitum (from P70, except one ketamine-treated rat) are indicated on the graph. (B) Ketamine-treated rats (KET) were compared with saline treated controls (SAL); there was a significant increase in the number of days required to reach learning criterion (unpaired t-test: t(12)=2.495, *P=0.028). (C) Number of days to reach criterion was similar for 3β—OH—treated rats compared with the β-cyclodextrin vehicle (CYCLO) treated animals (unpaired t-test: t(12)=0.079, P=0.938). (D) The cumulative percentage of rats reaching 100% as a function of days of trials was compared for the four groups. The acquisition rate of the ketamine-treated group (KET) was slower than that of saline controls (SAL) by Day 9 and remained substantially slower for the remainder of training. In contrast, rats in the 3β-OH group (closed squares) initially exhibited slightly faster acquisition than the vehicle control groups (SAL and CYCLO). However, by Day 16, their learning was identical to the learning curve of vehicle controls. Unlike rats in the ketamine group, all 3β—OH—treated rats completed the task in a manner practically indistinguishable from vehicle controls (n=7 rats per data point in panels A–D). (E) A percent-percent plot analysis shows that when only 28% of ketamine-treated rats (KET) had reached criterion, roughly half of 3β—OH—treated and vehicle control (SAL, CYCLO) rats had mastered the task. Whilst the learning curve of ketamine-treated rats remained flat, both 3β—OH—treated and vehicle-treated controls showed steady improvement enabling animals to reach criterion (100%), whilst about 85% of ketamine animals managed to master the task in the allotted time. The learning behaviour of 3β—OH—treated rats was similar to that of vehicle controls, resulting in a large gap (shaded area) in learning ability between ketamine-treated and 3β—OH—treated animals.

Fig 4

The effects of (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) on excitability of thalamic and subicular neurones. (A) Original traces from a representative thalamic neurone in control pre-drug conditions (black trace) portraying the loss of burst firing pattern as a response to both depolarising and hyperpolarising stimulus and in the presence of 3 μM 3β-OH (red trace). The inset shows concentrations of 3β-OH in rat brain achieved 5, 15, and 30 min after i.p. injection of 10 mg kg⁻¹ 3β-OH. (B) Application of 3 μM 3β-OH also significantly decreased the average rebound low-threshold spike (LTS), which underlies bursting in these neurones (paired t-test: t₄=3.14, P=0.035; n=5 neurones, three rats). (C) Original traces from a representative subicular neurone depicting control (black trace) and the effect of 3 μM 3β-OH (red trace) on the rebound firing pattern to a 200 pA hyperpolarising stimulus. On average 3 μM 3β-OH decreased the number of rebound action potentials from 1.41 (0.26) to 0.76 (0.21) (paired t-test: t(5)=3.81, P=0.013; n=6 neurones, three rats). (D) Original traces from a representative subicular neurone showing that 3β-OH reduced the amplitude of inward calcium currents (evoked using V_h of −90 mV and V_t of −40 mV). (E) Bar graphs showing averages from multiple experiments similar to panel (D) of this figure, which demonstrate that 3β-OH reduced the amplitude of calcium current by approximately 50% compared with baseline (pre-drug) control in the same cells (paired t-test: t₃=4.30, P=0.023; n=4 neurones, one rat), *P<0.05 vs baseline pre-drug conditions.

Fig 5

Effects of (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) on the evoked synaptic currents in the subiculum of P7−P9 rat pups. (A) Average original evoked inhibitory postsynaptic currents (eIPSC) traces in the absence (black trace) or presence (red trace) of 3 μM 3β-OH recorded using the paired-pulse protocol. The illustration depicts the placements of stimulatory and recording electrodes in CA1 and subiculum, respectively. (B) Column graphs show very little effect of 3 μM 3β-OH on the three parameters measured after the first pulse: eIPSC amplitude, decay time constant, and the paired-pulse (P2/P1) ratio (n=8 neurones, five rats). (C) Average original evoked excitatory postsynaptic currents (eEPSC) [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)] traces in the absence (black trace) or presence (red trace) of 3β-OH recorded using the same protocol described above. (D) Addition of 3 μM 3β-OH resulted in a significant reduction of AMPA-mediated eEPSC amplitude after the first pulse (paired t-test: t₄=3.75, P=0.020; n=5 neurones, two rats) without changing the decay time constant (t₄=0.93, P=0.403). The far-right bar graph shows that steroid had increased the paired-pulse ratio (t₄=5.28, P=0.006). (E) Average original eEPSC [N-methyl-d-aspartate (NMDA)-mediated] traces in the absence (black) or presence of 3β-OH (red trace) recorded using a paired-pulse protocol. (F) Bar graphs show minimal and non-significant effect of 3 μM 3β-OH on NMDA current amplitude and decay time constant after the first pulse, and on the paired-pulse ratio (n=5 neurones, three rats).

Fig 6

Effects of (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) on spontaneous GABA_A-mediated inhibitory postsynaptic currents (sIPSCs) in the subiculum of P7–P9 rat pups. (A) Original traces from a representative subicular neurone in the absence (black) or presence (red trace) of 3β-OH. (B) Cumulative probability plots for baseline (1682 events) and 3β-OH (2232 events) demonstrate longer inter-event intervals after the acute application of 10 μM 3β-OH. This finding is confirmed by the lower sIPSC frequency presented in the inset (paired t-test: t₈=3.32, P=0.011; n=9 neurones, five rats). (C) 3β-OH is devoid of effects on both amplitude (upper) and decay (lower) of sIPSCs, even at a relatively high concentration of 10 μM. (D) Original traces from representative subicular neurones in the control group (black trace), and groups pre-incubated with 3β-OH (red trace) or allopregnanolone (Allo, green trace). (E) Neurones pre-incubated with 10 μM 3β-OH (red) had longer inter-event intervals of sIPSCs than the control group (black), as shown by cumulative probability plots (5168 and 7341 events, respectively). The column graph in the inset shows lower frequency for groups treated with 3β-OH (n=19 neurones, nine rats) and Allo (green) (n=6 neurones, four rats) compared with control (n=19 neurones, nine rats). One-way analysis of variance (ANOVA): F_2,41=7.37, P=0.002; Tukey's post hoc test: P=0.017 and P=0.005, respectively. (F) The left bar graph shows that the amplitude of sIPSC events was not affected by different treatments (one-way ANOVA: F_2,41=2.44, P=0.100). Conversely, the right bar graph shows 500 nM Allo significantly prolonged the decay time constant (tau) compared with both the control and 3β-OH groups (one-way ANOVA: F_2,41=27.62, P<0.001; Tukey's post hoc test: P<0.001 vs both groups). *P<0.05, **P<0.01, and ***P<0.001 vs control group; ###P<0.001 vs 3β-OH group.