Although opioids are effective in treating pain, they cause serious side effects. The use of regional anesthesia, although effective in the perioperative period, may not be suitable if mobility and lack of numbness is desired. Hence, there is a clear need for novel pain therapies. Low-voltage activated (T-type) calcium channels (Ca_V3.2 isoform) could be a promising therapeutic target for the development of novel pain therapies. Indeed, our published findings suggest that novel neuroactive steroid (NAS) analogs that modulate the activity of Ca_V3.2 channels have unique anti-nociceptive properties. However, the concern with current NASs appears to be their hypnotic/sedative properties, thus potentially hindering the future development of NASs for novel pain therapies. Hence, we developed a new line of NASs that are effective blockers of neuronal Ca_V3.2 channels in pain pathways while having more favorable pharmacodynamic properties, i.e., lack of sedative/hypnotic side effects. We present two promising novel analogs of NASs—B372 ((3β,5α,17β)-3-Hydroxyandrostan-17-carbonitrile) and YX23 ((3β,5α,17β)-3-Methoxyestran-17-ol). Using an in vitro approach, we show that B372 and YX23 are effective in blocking Ca_V3.2 channels. Using an in vivo approach, we show that they are effective anti-nociceptives in wild-type but not Ca_V3.2 knock-out mice. Importantly, we show that they lack sedative/hypnotic effects.

Early-life general anesthesia (GA) may cause changes in socio-emotional behaviors in animals and autism spectrum disorder (ASD) in humans. The mechanisms behind GA-induced ASD symptoms are unknown. We investigate the mTOR activation as a potential cause of ASD. We assess ASD-like pathology after neonatal GA exposure to a volatile agent, sevoflurane, or an injectable GA, alphaxalone. We exposed male mouse pups on postnatal day 7 (PND7) to sevoflurane or alphaxalone (and their respective vehicles) for 6 h. We performed histomorphological analysis of caspase-3 activity in subiculum 2 h post-GA exposure and Western blot analysis of mTOR activation in hippocampus 24 h post-GA exposure. Spike firing in thalamic neurons was assessed at 4-6 weeks post-GA exposure. Behavioral tests for ASD-like features, including ultrasonic vocalization (USV) at PND8, nestlet shredding, marble burying, and 3-chamber social tests were conducted in adulthood. Sevoflurane, unlike alphaxalone, induced more nestlet shredding/marble burying compared to controls, and caused a shift away from the social preference and towards inanimate object. USV suggested a reduction in ultrasonic calls after sevoflurane, but not alphaxalone. The behavioral changes with sevoflurane were accompanied by an increase in caspase-3 activation, hyperactivation of mTOR, and an increase in neuronal firing compared to controls. The sevoflurane effects were largely reversed with rapamycin (a negative modulator of mTOR). Unlike sevoflurane, alphaxalone does not cause long-lasting ASD-type behaviors and does not affect the mTOR activation and histomorphology, suggesting that alphaxalone could be a safer alternative to sevoflurane.