Behavioral and mechanistic characterization of addictive properties of the classic anesthetic nitrous oxide and the anti-addictive effects of the natural compound cryptotanshinone

Abstract

Recreational use of nitrous oxide (N2O) has risen dramatically over the past decades. The pathogenesis of N2O abuse is poorly understood, and there is also a lack of effective medication for N2O use disorder. In this thesis, the tripartite objectives were proposed: (1) to determine the optimal conditions for N2O-induced conditioned place preference (CPP) model; (2) to verify whether N2O reward is associated with the regulation of molecular activities in ventral tegmental area (VTA)-nucleus accumbens (NAc) circuit; (3) to evaluate the therapeutic effects of cryptotanshinone (CTS) on N2O reward and its mechanism.

The optimization of N2O exposure conditions is based on orthogonal experiment. We examined three factors that influence the results of N2O exposure: N2O concentration, exposure duration, and exposure period. We divided C57 BL/6J mice by orthogonal design, and based on the results of CPP scores, the optimal conditions for N2O induction were as follows: Mice were exposed to 20% N2O for 4 consecutive days for 30 minutes each time. The obtained condition was further validated by CPP experiments, and the results showed that N2O could significantly improve the CPP scores under this condition.

Then, C57 BL/6J, Bdnf 2lox, DAT-cre and Vgat-Cre mice and SD rats were respectively received 20% N2O treatment to observe the molecular activities in VTA-NAc circuit. In C57 BL/6J mice, the repeated N2O exposure upregulated the expression of BDNF in VTA and its downstream mediators in NAc. Meanwhile, N2O improved synaptic plasticity in NAc of the mice. Microinjection of downstream mediator inhibitors into NAc of C57 BL/6J mice and VTA focal knockdown of BDNF in Bdnf 2lox mice both suppressed the N2O-induced CPP behaviors, and BDNF knockdown decreased the expression of downstream mediators in NAc. Moreover, N2O administration improved DA concentration in NAc and DAergic neuronal activity in VTA, but reduced GABAergic neuronal activity in VTA in mice. CPP behavior was weakened after microinjection of SCH23390 into NAc, and when combined with the BDNF knockdown intervention, this effect inhibited CPP behavior significantly more than either intervention. Rs-fMRI scan result showed that functional connectivity between VTA and NAc in rats was upregulated after N2O exposure. These results indicate that the rewarding properties of N2O are associated with activity modulation in VTA-NAc circuit.

CTS, isolated from salvia miltiorrhiza, has neuroprotective effects and can be rapidly metabolized into tanshinone IIA (TAN IIA) in vivo. After mice produced CPP targeting N2O, they were given a single daily gavage of 10, 20 or 40 mg/kg CTS and tested for CPP. The results showed that 20 and 40 mg/kg blocked N2O-induced CPP at third and fourth days after stopping supplying N2O, respectively. Meanwhile, CTS decreased N2O-induced activation of TrkB and D1R pathways in NAc. In addition, both CTS and TAN IIA reduced 7, 8-DHF plus SKF81297-induced activation of TrkB and D1R pathways in vitro.

Token together, N2O exerts its rewarding properties by increasing activities of VTA-NAc circuit, which is involved in the regulations of BDNF/TrkB pathway and DAergic neuronal activity. Moreover, CTS has potential to inhibit the rewarding effect of N2O.