PROJECT SUMMARY Many physiological processes and diseases exhibit intriguing rhythmic patterns such as cortisol secretion, body temperature, sleep/wakefulness, and epilepsy. With respect to epilepsy, it is well-known that seizures can occur in clusters, followed by long periods of seizure freedom, and many focal epilepsies exhibit approximately 24-h oscillations of seizure vulnerability. However, the mechanisms underlying periodicity in epilepsy are incompletely understood, and a detailed understanding of these mechanisms is important because this could result in more effective antiseizure interventions. Such interventions could be to increase the dose of antiseizure drugs or to change brain stimulation parameters during periods of heightened seizure susceptibility (i.e. chronotherapy), or to manipulate specific seizure-triggering chemistry perturbations using pharmacological, dietary, or even microbiological approaches. The long-term goal of the present project is twofold: first to understand the mechanisms of periodic vulnerabilities to seizures; second, to use this information to develop more effective and specific antiseizure therapies. The objective here is to identify circadian changes in the extracellular brain chemistry (metabolome) in humans with focal epilepsies and a translationally relevant rodent model of mesial temporal lobe epilepsy. To this end, we will track and quantify about 100 brain chemicals, including neurotransmitters, melatonin, adenosine, orexin, histamine, steroid hormones, and microbial metabolites. The central hypothesis is that numerous brain chemicals exhibit distinct circadian patterns associated with periods of increased seizure susceptibility, biological sex, brain region, neuropathology, sleep/wakefulness, light/dark cycle, antiepileptic drug therapy, and the host microbiota. The specific aim is to use in vivo brain microdialysis and mass spectrometry to explore changes in the extracellular brain metabolome during the circadian cycle in humans with epilepsy and a translationally relevant rodent model of mesial temporal lobe epilepsy. With respect to outcomes, this project is expected to identify multiple patterns of brain chemistry oscillations that correlate with periods of increased seizure vulnerability and many other biological parameters. The project will likely have a high positive impact because the identified rhythmic changes may be better understood and targeted to treat epileptic seizures more effectively than currently possible. Furthermore, the research is expected to markedly advance the field of chronobiology by revealing a complexity of novel circadian changes associated with key biological parameters such as sex, brain region, sleep/wakefulness, and the host microbiota. The proposal addresses the following 2014 NINDS Benchmark for Epilepsy Research: (IIIE) Identify, develop, and improve anti-seizure therapies that target (either alone, or in combination) novel or multiple seizure mechanisms.