Neonatal administration of a subanaesthetic dose of JM-1232(−) in mice results in no behavioural deficits in adulthood

In animal models, neonatal exposure of general anaesthetics significantly increases apoptosis in the brain, resulting in persistent behavioural deficits later in adulthood. Consequently, there is growing concern about the use of general anaesthetics in obstetric and paediatric practice. JM-1232(−) has been developed as a novel intravenous anaesthetic, but the effects of JM-1232(−) on the developing brain are not understood. Here we show that neonatal administration of JM-1232(−) does not lead to detectable behavioural deficits in adulthood, contrarily to other widely-used intravenous anaesthetics. At postnatal day 6 (P6), mice were injected intraperitoneally with a sedative-equivalent dose of JM-1232(−), propofol, or midazolam. Western blot analysis of forebrain extracts using cleaved poly-(adenosine diphosphate-ribose) polymerase antibody showed that JM-1232(−) is accompanied by slight but measurable apoptosis 6 h after administration, but it was relatively small compared to those of propofol and midazolam. Behavioural studies were performed in adulthood, long after the neonatal anaesthesia, to evaluate the long-term effects on cognitive, social, and affective functions. P6 administration to JM-1232(−) was not accompanied by detectable long-term behavioural deficits in adulthood. However, animals receiving propofol or midazolam had impaired social and/or cognitive functions. These data suggest that JM-1232(−) has prospects for use in obstetric and paediatric practice.

JM-1232(−) induces a small increase in apoptosis in the brain during development but is less than that induced by other anaesthetics. To investigate neurotoxic effects of JM-1232(−) on the developing mouse brain, we evaluated apoptotic cell death in the forebrain extracts using western blot analysis. Apoptotic cell death was detected by the presence of cleaved poly-(adenosine diphosphate-ribose) polymerase (PARP), a nuclear enzyme involved in apoptosis as described previously 5 . Compared with vehicle controls, administration of 1 mg kg −1 , 3 mg kg −1 , or 10 mg kg −1 of JM-1232(−) resulted in a small but significant expression of cleaved PARP 6 h after injection (Fig. 2). The amount of cleaved PARP expression was concentrationdependent (n = 4 or 5 mice for each dose; Kruskal-Wallis statistic = 8.33, p = 0.025, Kruskal-Wallis test; Fig. 2a).
Next, we evaluated the neurotoxic effects of midazolam in P6 pups. Consistent with the results from these studies 25 , we observed that midazolam (9 mg kg −1 ) increased significantly more cleaved PARP expression 6 h after the injection than the vehicle control mice, and that the amount of cleaved PARP expression was concentrationdependent (n = 5-7 mice for each dose; F = 30.3, p < 0.0001, one-way ANOVA; Fig. 2b).
western blot analyses showed that 6 h after injection propofol significantly increased expression of cleaved PARP more than the vehicle control mice, and the amount of cleaved PARP expression was concentration-dependent (n = 5 mice for each dose; F = 94.9, p < 0.0001, one-way ANOVA; Fig. 2c). When apoptosis levels at the respective dose of equivalent sedative effect were normalised to each anaesthetic's corresponding vehicle control, JM-1232(−) produced significantly less apoptosis than that produced by propofol or midazolam (JM-1232(−) [n = 5], midazolam [n = 7], propofol [n = 5]; F = 23.5, p < 0.0001, one-way ANOVA; Fig. 2d). All vehicle groups (solvents for JM-1232(−), midazolam, or propofol) were statistically indistinguishable in the apoptosis level among each other ( Supplementary Fig. S3 online).
Combined administration of JM-1232(−) and sevoflurane does not potentiate apoptosis compared with sevoflurane administration alone. Combined administration of inhalation and intravenous anaesthetics are used widely in clinical practice. A previous study indicated that sevoflurane in combination with propofol appears to induce more apoptosis than sevoflurane alone in the neonatal mouse brain, although the reason is unclear 28 . Sevoflurane is currently the most widely-used inhalation anaesthetic and is sometimes used in combination with propofol 29 . By contrast, thiopental, another intravenous anaesthetic, did not exacerbate the neurotoxicity of sevoflurane 28 . In the current study, we investigated whether apoptosis induced by sevoflurane is enhanced by JM-1232(−) injection. P6 mice were injected with 10 mg kg −1 of JM-1232(−) and JM-1232(−) induced lower levels of apoptosis when compared with those by propofol or midazolam. Apoptosis levels at the dose of equivalent sedative effect were normalised to apoptosis levels in the corresponding vehicle controls. Comparisons of the means among groups were performed using Kruskal-Wallis test followed by Dunn post hoc test (a) and one-way ANOVA followed by Tukey post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars are SEM. Full-length blots are presented in Supplementary Fig. 1 www.nature.com/scientificreports/ administered 2% sevoflurane by inhalation for 6 h, or were given each drug separately. Western blot analysis of forebrain homogenates of the single administration groups showed a slight increase in cleaved PARP level in the JM-1232(−) group but a large significant increase in the sevoflurane group (Fig. 3). The group that received the combination anaesthetics showed about the same level of apoptosis as the sevoflurane group alone. Twoway ANOVA revealed a significant main effect of sevoflurane exposure (n = 6 mice for each group; F = 86.0, p < 0.0001), but no significant main effect of JM-1232(−) administration (F = 2.0, p > 0.05). The interaction was not significant (F = 0.67, p > 0.05). Although 2% sevoflurane exposure significantly increased the expression levels of cleaved PARP more than the vehicle in the control group, the co-administration of JM-1232(−) and sevoflurane did not significantly alter the expression level compared with that of sevoflurane exposure alone (Fig. 3). Thus, JM-1232(−) does not enhance the apoptosis induced by sevoflurane.

JM-1232(−) has no detectable long-term effects on performance on cognitive function tests.
Previous animal studies indicated that neonatal exposure to general anaesthetics induces long-term adverse effects on cognitive functions 1,3 . Thus, we performed behavioural studies to compare any long-lasting effects of JM-1232(−) (10 mg kg −1 ), propofol (40 mg kg −1 ), and midazolam (9 mg kg −1 ) on the developing mouse brain when the mice reached adulthood (11-12 weeks old).
To assess short-term spatial working memory, mice were subjected to the Y-maze test. Memory performance of mice in the JM-1232(−) group was indistinguishable in adulthood from those in the control group (Fig. 4a). By contrast, mice in the propofol group made fewer correct choices (i.e., lower percentage alternation) compared with the adult mice in the JM-1232(−) group and midazolam group. One-way ANOVA and post hoc tests con- To assess associative memory, they were also tested for contextual/cued fear conditioning. Fear conditioning is a classical pavlovian model to assess associative memory for the pairing of a neutral CS (tone-cue) and the context with an aversive US (electric foot shock) 30 . Once this pairing is acquired, either the context or the tonecue alone induces the conditioned reaction, i.e. freezing behaviour (non-movement except for respiration).   www.nature.com/scientificreports/ p < 0.001, one-way ANOVA; Fig. 4b), mean freezing time for the propofol group was significantly less (i.e., worse memory) than the other groups. In the cued test (with cue; F = 3.07, p = 0.036, one-way ANOVA; Fig. 4c), mean freezing time in the propofol group was significantly less than the vehicle control group. These results indicate that neonatal administration of a single dose of JM-1232(−) or midazolam has minimal effects on some memory functions in adulthood, but propofol adversely affects memory function in adulthood.
Neonatal administration of JM-1232(−) has no detectable effect on sociability in adulthood. Mice are social animals and display clear social interaction behaviours 31 . We previously found that neonatal exposure to sevoflurane leads to social impairment in adulthood 3 . Sociability is a prominent behavioural aspect in autism-related behaviours. We used a rodent sociability test to assess a treated mouse's preference for another animate mouse (social) versus an inanimate mouse (stuffed toy mouse). Regardless of anaesthetic type administered in P6 mice, in adulthood they spent more time interacting with an animate real mouse than an inanimate fake mouse (p < 0.001 for all groups, Mann-Whitney test within group comparison; Fig. 5a). There were no significant differences among groups in time spent interacting with an animate target (vehicle control  Fig. 5a, right). The mean times spent interacting with the inanimate target were significantly longer for mice in the midazolam and propofol groups compared to that in the control group. These results indicate that neonatal exposure to midazolam or propofol leads to social impairment in The same set of mice tested in the Y-maze was tested in the fear conditioning tests. Comparisons of the means among groups were performed using one-way ANOVA followed by Tukey post hoc test (*p < 0.05, **p < 0.01). Error bars are SEM.  Fig. 5b). These results suggest that neonatal anaesthetic exposure leads to little or no disruption of olfactory sensation in adulthood, and thus, social impairment is not attributable to impairments of olfactory sensation. A restricted repetitive and stereotyped pattern of behaviour, expressed in self-grooming in rodents, was affected differently by the anaesthetics tested. This kind of repetitive stereotyped behaviour is another important behavioural aspect in autism 32 Fig. 5c). That for mice in the midazolam and propofol groups was significantly longer compared to that in the control and JM-1232(−) groups. These results indicate that neonatal administration of JM-1232(−) has minimal effects on the social functions and grooming behaviours assessed in these tests, but midazolam and propofol have detectable adverse effects. These results are further supported by the results of the three-chamber social approach task (see Supplementary Fig. S6 online).

Depression-like behaviours in adulthood are not apparent after neonatal JM-1232(−) administration.
To investigate whether neonatal administration of JM-1232(−) affects depression-like symptoms, we tested the treated and control mice on the tail-suspension test, a method for assessing depression-like symptoms 33 . In this test, we measured the latency to the first bout of immobility and the total time spent immobile during a 6-min trial. There were no significant differences among groups in the latency to the first immobil-  All groups showed a normal preference for the social target (left) over the inanimate target (right). However, mice receiving midazolam or propofol as neonates showed longer interaction times with the inanimate target than did control mice or mice receiving JM-1232(−) as neonates. Comparisons of the means among groups were performed using one-way ANOVA followed by Tukey post hoc test (*p < 0.05). Comparisons of the means within-group (social vs. inanimate) were performed using Mann-Whitney test (###p < 0.001). (b) Mean latency to find buried food in the olfactory test. All anaesthetic groups' latencies were statistically indistinguishable. (c) Mean grooming behaviour. Neonatal treatment with midazolam and propofol, but not JM-1232(−), led to longer time spent in adulthood grooming compared to controls. A comparison of the means among groups was performed using Kruskal-Wallis test followed by Dunn post hoc test (*p < 0.05, **p < 0.01). The same set of mice whose performance is presented in Fig. 4

Discussion
There is growing concern about the use of general anaesthetics in obstetric and paediatric practice 34 . Although extrapolating from animal models to humans can be fraught with difficulties, results from animals suggest that repeated or lengthy use of general anaesthetic during surgery in children younger than 3 years may affect brain development, which can be manifested later in adulthood as cognitive problems. The mechanisms underlying the neurotoxicity of general anaesthetics in the developing brain are not yet fully understood, and thus, effective alternative anaesthetic is still lacking. Assessing potential new anaesthetics in animal models is the first step in determining whether a particular anaesthetic can be safely used in clinical setting.
Although neurodevelopmental age equivalencies between mice and humans cannot be specified with precision, our decision to use 6-d-old pups for the present study was based on the assumption that this neurodevelopmental age in the mouse is equivalent to the critical period of the human age 35 . The critical period is a distinct time-window in the neonatal stage when animals display elevated sensitivity to certain environmental stimuli, and particular experiences can have long-lasting and profound effects on behaviours. The critical period of neurobehavioral development is a time of learning opportunity but also of vulnerability for interruption: interruption during the critical period can cause irreversible and permanent problems in the brain. It is well known that the closure of one eye (monocular deprivation) of a kitten during the critical period results in loss of visual acuity in the deprived eye despite no physical damage to the eye itself 36 . Studies about anaesthetic-induced toxicity in brain development indicate that anaesthetic-induced apoptosis is the greatest if exposure occurs at P6-P7 1,3,37 , with little or no increase in apoptosis at P14 in rodents 37 . Furthermore, neonatal exposure of pups to anaesthetics at P6-P7 could cause behavioural impairments later in adulthood 1 , including social deficits similar to those seen in autistic spectrum disorder 3 . Thus, there may be a critical period of vulnerability for the exposure to anaesthetics  www.nature.com/scientificreports/ with the peak at P6-P7 and the critical period would be closed before P14 in rodents.In the current study, we found that a single injection of JM-1232(−) at subanaesthetic dose resulted in a small increase in apoptotic cell death in the developing mouse brain. However, the level of apoptosis resulting from JM-1232(−) was significantly lower than that resulting from dose-equivalent sedative effects of propofol or midazolam. Immunohistochemical analysis confirmed that the number of cells with activated (cleaved) caspase-3 + (AC3 + ) significantly increased in mice treated with anaesthetics at P6 compared to vehicle controls ( Supplementary Fig. S4 online). However, the number of AC3 + cells in the JM-1232(−) group was significantly lower than that of the propofol or midazolam groups ( Supplementary Fig. S4 online). Distribution patterns of degenerating neurons in all three types of anaesthetics are similar to the pups with sevoflurane exposure in our previous studies 3, 38 ; the increased apoptosis is most robust in the retrosplenial cortex, subiculum, neocortex, and thalamus in the brains of pups with anaesthetic exposure (Supplementary Fig. S4 online). Most importantly, we did not detect any behavioural deficits when mice reached adulthood after neonatal JM-1232(−) injection, in contrast to the other widely-used general anaesthetics. However, a causal link between apoptosis appearing immediately after anaesthesia and behavioural deficits manifested later in life remains unknown.
Our results indicate that toxicity of JM-1232(−), midazolam, and propofol are different, although the underlying mechanism is unknown. General anaesthetics modulate specific ligand-gated ion channels, principally GABA A receptors and/or N-methyl-d-aspartate (NMDA) receptors, the latter being a subtype of glutamate receptors. Several studies have demonstrated that NMDA receptor-mediated and GABA A receptor-mediated responses both have adverse effects on the developing brain 4,7,10,39 . A previous study suggested that apoptosis is synergistically potentiated when both NMDA and GABA A receptors are simultaneously affected in the developing brain 4 . It is well known that exposure of the foetus to ethanol during critical periods of development induces foetal alcohol syndrome. Ethanol acts through both NMDA and GABA A receptors. In this context, although propofol acts mainly by binding to GABA A receptors, it also acts, to a lesser extent, by binding to NMDA receptors 20 . JM-1232(−), on the other hand, binds to GABA A receptors, and it barely inhibits glutamine receptors 40 . Thus, the different effects of propofol and JM-1232(−) on the developing brain might result from different effects of the two drugs on the NMDA receptor.
Propofol is sometimes co-administered with sevoflurane being one of the common inhalation anaesthetics 29 . Sevoflurane binds to GABA A receptors similar to many intravenous anaesthetics 41,42 . A previous study reported that 3% sevoflurane in combination with 10 mg kg −1 propofol induces a more robust apoptosis than sevoflurane alone in the neonatal mouse brain 28 . In contrast, our results demonstrated that co-administration of 2% sevoflurane with JM-1232(−) (10 mg kg −1 ) did not significantly increase the level of apoptosis compared with 2% sevoflurane alone. These differences also suggest that the mechanisms of toxicity of JM-1232(−) and propofol likely differ.
Previous studies showed that exposure of the immature brain to benzodiazepine can result in cognitive alterations lasting long after the cessation of benzodiazepine exposure, although results of these studies are inconsistent [43][44][45] . This inconsistency probably relates to differences in the schedules of drug administration, different ages among those tested, different drugs, and differences in tests. In consistent with our results, Mikulecka et al. reported that neonatal exposure to rats (from P7 until P11) with therapeutically relevant doses of clonazepam, a classic benzodiazepine, lead to disturbances of cognitive-like behaviour 46 , and social deficits later in adulthood 47 . Although both midazolam and JM-1232(−) act by binding to the benzodiazepine site of GABA A receptors 16 , only mice administered midazolam as neonates exhibited social impairments and enhanced grooming behaviour during adulthood in the current study, even though both anaesthetics were given at sedativeequivalent dosages. While the mechanisms underlying the neurotoxicity of midazolam and JM-1232(−) are not well understood, it may be noteworthy that the molecular structures of the two are significantly different (i.e., benzodiazepine structure of midazolam vs. nonbenzodiazepine structure of JM-1232(−)).
In the current study, mice received only a single injection of JM-1232(−) at P6. Previous reports suggest that repeated administration of general anaesthetics may cause more severe neurotoxicity compared to a onetime administration 27 . Thus, our results might have been different if we had exposed mice to multiple episodes of JM-1232(−).
Because of the animal-model caveat, the relevance of our findings in mice to human neonates is unknown. Thus, it is too early to say definitively whether JM-1232(−) has the same mild effect in humans. However, it is reasonable to propose the idea that JM-1232(−), or its derivatives, could be more favourable as an obstetric and paediatric anaesthetic, since its neurotoxic effects are minimal on the developing mammalian brain at least one instance of an animal model. With better knowledge in the future, we would be able to anaesthetize pregnant mothers and infants safely. Until then, we would be to select anaesthetics that are the least harmful.

Methods
Animals. All experiments were conducted according to the institutional ethical guidelines for animal experiments of the National Defense Medical College (Tokorozawa, Japan). The experimental protocol was approved by the Committee for Animal Research at the National Defense Medical College (approval number: 17074). This study was carried out in compliance with the ARRIVE guidelines (http:// www. nc3rs. org. uk/ page. asp? id= 1357).
Inbred, C57BL/6 male mice were used in this study (CLEA Japan, Inc., Tokyo, Japan). Age of the mice ranged from P6, when anaesthesia was administered, to 11-12 weeks of age, when behavioural tests were administered. The mice were housed under standard laboratory conditions, with a 12-h light/dark cycle and a room temperature maintained at 23 ± 1 °C. The mice were given access to water and food ad libitum.
In total, n = 314 mice were used in the study. However, one pup in the midazolam group was died unexpectedly prior to the behavioural tests, preventing analysis, and another pup was died during the anaesthesia with Y-maze test. The Y-maze test evaluates spatial working memory 50 and was administered as described previously 51 . Briefly, each mouse was placed in the centre of the Y-maze, after which it was allowed to freely explore the maze for 8 min. The sequence of arm entries and total number of arms entered were recorded. An arm entry was counted when all four limbs of the mouse were completely on an arm. The percentage of alternations was the number of triads containing entries into all three arms divided by the maximum possible number of alternations (total number of arm entries minus 2) × 100. Fewer correct choices can indicate poorer spatial working memory.
Fear-conditioning test. The fear-conditioning test assess associative memory 30 and was performed as previously described 49 . Briefly, the conditioning trial for contextual and cued fear conditioning consisted of a 5-min exploration period followed by three conditioned stimulus-unconditioned stimulus pairings (CS-US), each separated by a 1 min intertrial interval Olfactory test. The olfactory test was conducted as described previously 49 . Briefly, on the first day of the test, mice were habituated to the flavour of a novel food (blueberry cheese). After 48 h of food deprivation, the mouse was placed in a clean cage, and the time elapsed for the mouse to find the buried piece of blueberry cheese was measured. The blueberry cheese was buried under 2 cm of test cage bedding.
Tail-suspension test. The tail-suspension test, which evaluates depression-like symptoms, was administered as described previously 35 . Briefly, a mouse was suspended from the edge of a desk by attaching its tail to the desk with adhesive tape. The adhesive tape was placed approximately 5-10 mm from the tip of the tail. The suspended animal was 600 mm away from the floor. The total duration of immobility (i.e., lack of movement with the head pointed downward) and the latency to the first episode of immobility were measured during a 6 min test period.
Forced-swim test. The forced-swim test, another assay that measures depression-like symptoms in rodents, was administered as described previously 35 . Briefly, a mouse was placed in a cylinder (25 cm diameter, 46 cm depth) filled two-thirds with water (25 ± 1 °C) for 6 min. The mouse could not escape from the cylinder, and its feet could not touch the bottom of the cylinder. Naïve mice typically swim in the water to search for an escape route from the water. This test measures the time spent swimming versus the time spent floating. Swimming behaviour was defined as active horizontal movement through the water that exceeds that necessary to merely maintain the head above the water. It is a proxy measure to assess 'hopefulness' . Floating behaviour was defined as lack of movement beyond what is necessary to maintain balance and to keep the nose above water; it was measured as a proxy variable of 'hopelessness' . After each trial, the mouse was lightly towel-dried and introduced back into its home-cage. The water in the cylinder was changed between each animal.
Statistical analysis. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc, La Jolla, CA USA). Sample size was defined according to previous studies 38,49,53,54 . To obtain ED 50 value, we used a variable slope model (nonlinear fit [agonist] versus normalized response-variable slope, GraphPad Prism 8).
Statistical comparisons between the means of each treatment group were performed using t-test, one-way analysis of variance (ANOVA), or two-way analysis of variance (ANOVA) followed by a Tukey post hoc test, when the assumption of normality was satisfied. When the assumption of normality was not satisfied, comparisons were made using Mann-Whitney test or Kruskal-Wallis test, followed by a Dunn post hoc test. Data are presented as means ± standard error of the mean (SEM). The ED 50 values were presented with 95% CI.

Data availability
Data are available from the corresponding author upon a reasonable request and with permission of Dr. Yasushi Satoh.