Hundreds of millions of patients are exposed to general anesthetics (GAs) each year, making the heritable effects of GAs a public health issue of paramount importance. The need to investigate the heritable effects of GAs and develop preventative therapies is also indicated by an unprecedented rise, particularly in industrialized countries, of neurodevelopmental disorders. The origin of most of these disorders is unknown and many are more common in males. To form the basis for clinical studies on this topic, the proposed preclinical project will test the following hypotheses regarding the mechanisms, therapeutic tools, and biomarkers pertaining to heritable effects of GAs: 1) GA-induced secretion of the steroid stress hormone corticosterone (CORT) is essential in the initiation of epigenetic changes in parental germ cells (F0 generation) and, by extension, abnormalities in offspring (F1 generation); 2) both GABAergic GA-induced impairment of the K+-2Cl- (KCC2) Cl- exporter, resulting in impairment of inhibitory GABAAR signaling, and an increase in CORT secretion are required for GA-induced F0 neurobehavioral defects; and 3) F1 males are more vulnerable because F0 GAs act via modification of male- specific F1 brain masculinization. Aim 1: Determine the roles of KCC2 and CORT in the initiation of intergenerational effects of sevoflurane (SEVO). Four clinically used GAs with partially overlapping mechanisms of action, SEVO, propofol, ketamine and etomidate, will be used as pharmacological tools to determine the roles of KCC2 and CORT. The F0 rats will be exposed to GAs on postnatal day (P) 56, P58, and P60 and mated on P85 to generate offspring. The F0 and F1 rats will be evaluated in the elevated plus maze, prepulse inhibition of startle*, Morris water maze, fear-potentiated startle, and forced swimming test and by assessing resting and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity*. Genome-wide DNA methylation in F0 and F1 germ cells and hippocampi and RNA-seq in F0 and F1 hippocampi and peripheral blood monocytes* will be used to gain insight into epigenomic and transcriptomic mechanisms (* = will be evaluated as potential biomarkers for future human studies). Aim 2: Determine whether shorter parental SEVO exposure that is not sufficient to induce F0 neurobehavioral effects can induce F1 effects, and determine the roles of Cl- transporters and glucocorticoid receptors (GRs). Hypotheses: Shorter F0 SEVO exposure is sufficient to upregulate the F0 HPA axis and reprogram the germline, inducing F1 defects. KCC2 enhancement or Na+-K+-Cl- (NKCC1) and GR inhibition have therapeutic effects. Aim 3: Determine the mechanisms of male-biased intergenerational effects of parental exposure to SEVO. Hypotheses: F1 males are more vulnerable because F0 SEVO affects testosterone (T)- regulated brain masculinization. Because T acts through 17β-estradiol (E2) at estrogen receptor α (ERα) and via T-activated androgen receptors (ARs), we hypothesize...