Series 1
In series 1 we assessed the effects of acute subanesthetic doses of Xe on normal rats in a battery of behavioral tests. Of note, this is the first study in which a short-term exposure to Xe gas was used to evaluate such effects. In all of the previous studies animals were treated with 25–70% Xe for at least 20 min and up to 5 h [
22,
24,
38‐
42]. Importantly, pharmacokinetic analysis of exposure to 50% Xe has shown that the maximal concentration of Xe in the rats’ brain is reached within the 1st min of exposure [
43]. These data suggest that even a short-term exposure to Xe can affect neurotransmission.
Our results suggest that Xe-exposed rats showed no sign of sedation as there was no decrease in horizontal locomotion and grooming activity in the OF test. Besides a measure of locomotor activity, the OF test measures other factors such as exploratory drive and anxiety-like behavior. Some outcomes, particularly defecation, center time, and activity within the first 5 min, may also measure some aspects of emotionality [
44]. The decrease in rears, center entries and time spent in the center of a maze may suggest a suppression of exploratory motivation or enhanced emotionality evoked by novel conditions in adolescent rats after acute inhalation of Xe.
Also, in the EPM test we have observed a trend towards decline in the time spent on the distal edges of the open arms and in the number of head-dips in Xe-treated animals. There were no changes in anxiety-related behaviors between “Control” and “Xe” groups. These results suggest a modulatory effect of Xe inhalation on proclivity of risk-taking behavior and no effect on anxiety level of adolescent rats.
In the FS test we observed a tendency towards decreased time spent immobile and increased latency to first immobilization. The reduction in immobility is usually identified as an antidepressant-like effect [
44]. The effects of acute Xe administration resulted in tendency to reduce behavioral despair during forced swimming test.
In addition, an increased latency to leave the closed arm with no changes in other parameters in the OM suggests reduction of exploratory motivation in Xe-exposed rats. The OM was conducted 5 days after the last Xe inhalation, suggesting the prolonged effect of previous Xe exposures. Based on these results we can conclude that 10 min of 25% Xe inhalations is sufficient in modulating behavior of adolescent animals. Wherein, the exposure to Xe didn’t alter either “normal” locomotor activity nor the “normal” anxiety level of rats in the open field, elevated-plus and elevated O-maze tests. At the same time, Xe administration resulted in reduced exploratory motivation and/or emotionality evoked by novel conditions in OF and OM, and slightly decreased the risk-taking behavior in EPM. In addition, Xe has shown ability to reduce behavioral despair of rats in FS. The decreased exploratory motivation in OM suggests delayed, long-term effects of Xe inhalation. There have been no previous studies on behavioral changes in healthy rats after Xe treatment. The behavioral modulatory effects of Xe which was shown in series 1 are probably related to its generalized effect on excitatory/inhibitory balance within the CNS through NMDA inhibition and/or TREK-1 activation [
45,
46]. We propose that these effects of Xe might be translated into the treatment of psychoemotional disorders.
Series 2
The most important finding of this study is demonstration of improved social behavior of VPA-exposed rats after a single inhalation of Xe (25%, 10 min). Prominent inhibitory effect of Xe on NMDA receptors makes this gas an attractive modality for studying pathological conditions involving these receptors. Rats prenatally exposed to VPA show increased NMDAR levels, enhanced NMDAR-dependent LTP, and hyperconnected local neocortical circuits [
47,
48]. In the current work, aggressive behavior as well as social anxiety of rats treated with VPA from pnd 6 to pnd 12 at the dose of 150 mg/kg was significantly reduced by acute Xe administration. These data suggest beneficial effect of subanesthetic short-term exposure to Xe and its possible implication in the treatment of psychoemotional disorders such as ASD.
To assess the outcomes of postnatal VPA exposure and the subsequent Xe inhalations in early life we have conducted tests for sensorimotor development of rats. The negative geotaxis test in neonatal rodents is a measure of sensorimotor function, strength and stamina [
49]. The gait test is used to assess integrity of the cerebellum and of the muscle tone [
50]. Such complex behaviors involve both excitatory and inhibitory pathways which can be affected by endogenous and exogenous substances. Xe inhalation led to an improved integrative sensorimotor response in the negative geotaxis test, but not in gait reflex in healthy animals. It must be noted that raised quadruped posture and locomotion is fully developed in rats by day 16 [
35]. It is plausible that Xe affects the development of sensorimotor function but has no effect when it’s fully developed. The possible mechanism of action of Xe may be in modulation of signal transduction through regulation of NMDA receptors. There was no significant effect of postnatal VPA treatment on sensorimotor development of rat pups and no effects of Xe administration in valproate animals. There is a paucity of information on developmental and behavioral outcomes in postnatal VPA models. It has been previously reported that VPA exposure on embryonic day 12.5 didn’t alter the performance of rats in negative geotaxis test [
51]. Also, it was shown that exposure to VPA from pnd 6 to pnd 12 at the dose of 150 mg/kg didn’t affect physical development of rats but led to the disruption of motor skills involved in object manipulation [
52]. Other studies using prenatal or postnatal VPA exposure have reported its negative effects on sensorimotor development of rodents, but not in all paradigms [
51,
53‐
55].
In series 1 we observed a decrease in exploratory motivation and/or emotionality of rats after exposure to Xe. In series 2 we have observed an effect of Xe administration similar to that in the previous experiment, showing a decrease in locomotion during the first 2 min of the experiment in Xe-exposed healthy and Xe-exposed VPA animals as well as a trend towards a decrease in rearing in “Xe” group. The performance in OF test during the 1st min of experiment measures a reaction to novelty rather that general activity [
44]. In support of “low exploratory motivation” theory it should be noted that previous studies reported no sedating effects of xenon after 30 min treatment with 50% Xe/50% O
2 gas mixture [
41]. In our study the exposure to valproate from pnd 6 to pnd 12 didn’t affect animal performance in the OF test. The data on motor activity of rodents in VPA models of ASD’ are quite conflicting. Both increased [
53,
56,
57] and unchanged [
54,
58] locomotion in valproate-treated animals independent of dose and prenatal/postnatal administration of VPA have been reported. A recent study reported a 2% frequency of co-occurrence of attention-deficit hyperactivity disorder in children with ASD [
59], making this comorbid diagnosis rather uncommon in ASD.
The effects of postnatal VPA administration and Xe exposure on exploratory behavior and anxiety were studied in the EPM. Anxiety is the most common comorbid psychiatric symptom in children with ASD, with prevalence rate reaching 40% [
60]. Anxious behaviors are also reported in the studies using VPA model of ASD in EPM [
53,
55,
58,
61‐
63]. We should note that in our study “VPA” group of animals has shown a tendency towards anxious behaviors, whereas “VPA + Xe” group has had a pronounced anxiety-like behavior in elevated plus maze. After acute inhalation of Xe healthy animals didn’t differ from controls. Furthermore, in series 1, Xe has shown potency to reduce risk-taking behavior in adolescent rats, but in series 2 these differences were lost probably due to the reduction of the sample size. Thus, it remains unclear whether Xe inhalation enhanced anxiety-like behavior in VPA-exposed rats. Moreover, some authors propose potential effectiveness of Xe inhalations in treatment of anxiety disorders [
32,
64]. Still, further studies broadening an understanding of anxiety-like behavior after Xe inhalation in VPA model of ASD’ may be warranted.
The most prominent feature of autism is social impairment. Most of the studies of behavior in VPA model revealed some, but not all impairments of social interaction [
55,
58,
65]. In our study we have shown that postnatal exposure to VPA results in increased aggression towards unknown age-matched animals which was expressed as the increase in the number of pinning, pouncing and sniffing. It is plausible that such reaction is associated with negative emotions towards a stranger. In the study of Kataoka et al. [
66], female but not male mice prenatally exposed to VPA spent more time sniffing age-matched animal, without affecting allogrooming or aggression. Aggressive behavior is frequent yet poorly understood in children with ASD [
67]. Also, we have observed an increase in latency to leave starting area in social novelty test. We suggest that such reaction was caused not only by the new conditions, but also by the acoustic and olfactory signaling from dam and unknown female, which resulted in the so-called “social anxiety” in adolescent rats. It has been estimated that 40% of ASD patients have at least one anxiety disorder, with social anxiety being one of the most common among them (17%) [
60]. Xe administration prior to testing resulted in normalization of aggressive behavior as well as anxiety in VPA-exposed rats. There was no change in social behavior in healthy animals after Xe inhalation. It is plausible that Xe’s modulating effect on excitation/inhibition in CNS occurs mainly through its inhibitory effect on excitatory neurotransmission per se.
In the current study we used the forced swim test as a behavioral paradigm that has been found useful in assessing possible antidepressant activity of drugs [
44]. In series 1 we have observed a tendency towards antidepressant effects of Xe, which was not replicated in series 2, probably due to a smaller sample size. Though, we have shown a trend towards increase in climbing activity in both VPA-exposed groups, with significant rise of struggling behavior in the second half of the testing in VPA group in comparison with Control group. During testing, animals of all experimental groups have shown a reduction in climbing activity over time. Because struggling behavior consumes a lot of energy, animals tend to employ energy conserving behavior during testing, such as active swimming and passive floating as a successful coping strategy [
68]. The increased climbing in the second half of the experiment may indicate an impaired inhibitory reaction (habituation) which is normally formed during the long testing session. This may also indicate an impaired decision-making in VPA animals with increased probability of earlier exhaustion. Pre-exposure to Xe didn’t affect the overall climbing activity but reduced such behavior to control levels in the second half of the experiment. It is plausible that reduced climbing is a beneficial effect as it strategically helps to conserve energy in a stressful environment. There is paucity of information on behavioral changes in FS test in VPA models of autism with one previous study showing increased depressive tendencies in rats which were prenatally exposed to VPA [
55].
Valproic acid model may replicate some but not all symptoms of ASD. The most prominent feature of autism is social impairment. In our study VPA treatment from pnd 6 to pnd 12 at a dose of 150 mg/kg led to increased aggression and anxiety. These effects are the most commonly observed signs of autism in clinical practice. We observed neither any physical or sensorimotor impairment in animals in early infancy, nor locomotor disturbances or depressive-like behavior in adolescence. There is evidence that behavioral and developmental outcomes in VPA model of ASD depend on dose and timing of exposure to valproate. A time frame from pnd 6 to pnd 12 reflects sensitive period of functional development of the rats’ brain during which the processes of proliferation, synaptogenesis and myelination are ongoing. Moreover, at pnd 6–8 a peak in the expression level of NMDA receptors occurs in rodents’ brain and this increase is presumably necessary for the developing brain because activation of glutamate system plays a substantial role in the morphogenesis and development of CNS plasticity [
69]. Thus, postnatal exposure to VPA may lead to a delayed long-lasting effect mimicking ASD [
33].