Endocannabinoid Release Calms Epileptic Seizures but Also Leads to Adverse After-Effects

Studies in live mice by Stanford University School of Medicine investigators have shown how epileptic seizures trigger the rapid synthesis and release of an endocannabinoid compound, 2-arachidonoylglycerol, or 2-AG, which is mimicked by marijuana’s most psychoactive component. But while the seizure-triggered release of 2-AG in the brain has the beneficial effect of damping down seizure intensity, the rapid breakdown of 2-AG into a molecule that is a building block for inflammatory prostaglandins effectively trips off a cascade of biochemical reactions culminating in blood-vessel constriction in the brain and, in turn, the disorientation and amnesia that typically follow an epileptic seizure. The researchers suggest that their discoveries could guide the development of drugs that both curb the strength of a seizure, but also dampen these after-effects.

Reporting on their findings in Neuron (“In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity”), co-senior author Ivan Soltesz, PhD, professor of neurosurgery, and colleagues at Stanford and at other institutions in the US, Canada, and China, concluded, “…the finding that excessive 2-AG feeds this vasoconstriction pathway supports two opposing roles (anti-seizure versus pro-hypoxia) that are fundamental to our understanding and treatment of epilepsy.” Co-senior author of the paper is G. Campbell Teskey, PhD, professor of cell biology and anatomy at the University of Calgary in Alberta, Canada. The study’s lead author is Jordan Farrell, PhD, a postdoctoral scholar in the Soltesz group.

Epilepsy affects about one in every hundred people. Epileptic seizures can be described as an electrical storm in the brain, which typically begin at a single spot, where nerve cells begin repeatedly firing together in synchrony. The hyperactivity often spreads from that one spot to other areas throughout the brain, causing symptoms such as loss of consciousness and convulsions. It’s typical for the person experiencing a seizure to need tens of minutes before becoming clearheaded again.

The majority of epileptic seizures originate in the hippocampus, a brain structure buried in the temporal lobe, explained Soltesz, who is the James R. Doty Professor of Neurosurgery and Neurosciences. The hippocampus plays an outsized role in short-term memory, learning and spatial orientation. Its ability to quickly adopt new neuronal firing patterns renders it especially vulnerable to glitches that initiate seizures.

2-AG is an endocannabinoid (eCB), a member of a family of short-lived signaling substances that are the brain’s internal versions of the psychoactive chemicals in marijuana. 2-AG and these plant-derived psychoactive chemicals share an affinity for a receptor, known as the cannabinoid type 1 receptor (CB1). This is the most abundant G protein-coupled receptor in the brain, and plays important roles in physiological and pathophysiological states, including epilepsy, the authors explained.

Endocannabinoids are understood to play a role in inhibiting excessive excitement in the brain. When excitatory neurons, secreting chemical “go” signals, exceed a threshold, they induce the production and release of endocannabinoids, which bind to CB1 on an excitatory neuron and act as a brake, ordering that neuron to chill out a little.

2-AG and anandamide (AEA), are the main natural lipid ligands for CB1. However, the relative contributions of AEA and 2-AG are still debated, at least in part because of the limitations of current in vivo experimental techniques.

While smoking marijuana floods the entire brain with the relatively long-lasting psychoactive cannabinoid compound, THC, the brain’s natural endocannabinoids are released in precise spots under precise circumstances, and their rapid breakdown leaves them in place and active for extremely short periods of time, said Soltesz, who has been studying the connection between endocannabinoids and epilepsy for decades.

But because endocannabinoids are so fragile and break down so quickly, until recently there was no way to measure their fast-changing levels in animals’ brains. “Existing biochemical methods were far too slow,” he pointed out. “Until recently, our understanding of eCB dynamics in vivo has been limited by the inappropriately low spatiotemporal resolution of conventional biochemical approaches,” the team further stated. “Thus, a fundamental understanding of how eCB levels fluctuate with neural activity is missing.”

For their newly reported research, Soltesz and his associates monitored split-second changes in levels of 2-AG in the hippocampus of mice during periods of normal activity, like walking or running, and in experiments in which brief seizures were induced in the hippocampus.

“There have been lots of studies providing evidence for a connection between seizures and endocannabinoids,” Soltesz said. “What sets our study apart is that we could watch endocannabinoid production and action unfold in, basically, real time.”

The reported study had its start when Soltesz learned of a new endocannabinoid-visualization method invented by study co-author Yulong Li, PhD, a professor of neuroscience at Peking University in Beijing. The method involves the bioengineering of select neurons in mice so that these neurons express a modified version of CB1 that emits a fluorescent glow whenever a cannabinoid binds to the modified endocannabinoid receptor. The fluorescence can be detected by photosensitive instruments.

Using this new tool, called GRAB-eCB2.0, the scientists could monitor and localize sub-second changes in fluorescence that correlate with endocannabinoid levels where that binding was occurring. By blocking enzymes critical to the production and breakdown of different endocannabinoids, the researchers demonstrated that 2-AG alone is the endocannabinoid substance whose surges and rapid disappearance track neuronal activity in the mice. Several hundred times as much 2-AG was released when a mouse was having a seizure, compared with when it was merely running in place.  Anandamide’s name is derived from the Sanskrit word for “bliss.”

“This previously undetected activity-dependent surge in levels of 2-AG downregulates excitatory neurons’ excessive rhythmic firing during a seizure,” Soltesz said. The researchers were also able to rule out the involvement of the endocannabinoid, anandamide, which many neuroscientists and pharmacologists had assumed was the active substance.

However, they found, 2-AG is almost immediately converted to arachidonic acid, a building block for inflammatory compounds called prostaglandins. The researchers’ studies then showed that the ensuing increase in arachidonic acid levels resulted in the build-up of a particular variety of prostaglandin, which causes constriction of tiny blood vessels in the brain where the seizure has induced that prostaglandin’s production, cutting off oxygen supply to those brain areas.

“These data provide evidence that 2-AG serves as the dominant activity-dependent endocannabinoid in the hippocampus and is coupled to physiological neural activity in a precise spatiotemporal manner,” the team stated. “Mounting evidence supports that the eCB system is a prime target for seizure control, and these data provide clear in vivo evidence that 2-AG is produced in an activity-dependent manner and is necessary to suppress seizures. However, this previously undetected surge of 2-AG during seizures comes at the cost of producing extremely high levels of PGE2 that leads to prolonged hyperperfusion/hypoxia.”

Oxygen deprivation is known to produce the cognitive deficits—disorientation, memory loss—that occur after a seizure, Soltesz said. “A drug that blocks 2-AG’s conversion to arachidonic acid would kill two birds with one stone. It would increase 2-AG’s concentration, diminishing seizure severity, and decrease arachidonic acid levels, cutting off the production of blood-vessel-constricting prostaglandins.”