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MacEwan University researchers test effects of cannabis terpenes on zebrafish

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MacEwan University’s neuroscience research teams are pioneering work on the effects of cannabis terpenes on zebrafish. Terpenes are what give many plants their smells and cannabis terpenes may have beneficial medicinal properties. These teams are some of the first in the world to begin testing on the super-class terpenes and are finding exciting results. MacEwan researchers have found that there are significant changes to fish behaviour when the fish
are dosed — the next step is observing how this affects their brains.

Cannabis is currently a hot topic for research with three popular areas of interest. “You got the CBD, you got the THC, and you have the terpenes,” explains Ryan Verbistky, a student in the bachelor of science program with a major in psychology at MacEwan. He is doing his independent study on the effects of terpenes on zebrafish. MacEwan teams have tested different terpenes like beta-myrcene, linalool, limonene, alpha-pinene, and more. Many of these terpenes are also found in essential oils, flowers, and plants.

Terpenes are responsible for the flavours and smells of cannabis but could also have medicinal and therapeutic effects. Dr. Trevor Hamilton, an associate professor in psychology, defines them as “aromatic compounds present in plants and flowers. So there’s over 30,000 different types of terpenes and about 400 types present in cannabis.” The terpenes present vary between cannabis plants which give a lot of different aroma profiles to cannabis.

A 2022 paper that was published by one research team, Andréa Johnson, Alycia Stewart, Ismaeel El-Hakim, and Trevor J. Hamilton, tested the effects of the terpenes beta-caryophyllene and alpha-pinene. Alpha-pinene is a very common terpene and it is found in sage, pine, lavender, and beer. In this study, they tested alpha-pinene as a whole and as its negative and positive forms (enantiomers). The results showed that as a whole, this terpene did not have much effect on the zebrafish anxiety level. But in its separated forms, it did show decreased anxiety that has led to current research.

The process for testing the effects of terpenes on the fish is relatively the same for different studies. A zebrafish would be taken from the regular fish tanks and put into beakers containing the terpene under question. The researchers calculate different dosages of each terpene and administer it to fish one-by one. Once the fish are dosed, they are put into an open field test, which is a cylinder tank made of polyurethane. Next is the novel object approach test which involves placing a Lego figurine into the centre of the tank. Alycia Stewart, co-author of the published paper, explains the steps: “I would only dose them for 10 minutes then they would enter the study — enter the open field test for 10 minutes and then do the novel object approach test for 10 minutes — and then… they’d get put into a container or into a tank to (have some time to) chill out,” Stewart explains.“They wouldn’t be able to be used for further testing. That’s the cycle that I got to use in my experiment, that they (the fish) went through.” Above the arena is a video camera connected to a motion tracking software system used to record and analyze the data. This way, the researchers can track how the fish respond to the terpenes based on tests of boldness, locomotion, and anxiety-like behaviour.

To track the anxiety levels of fish, they divide the tank into several sections that start from the outside of the tank towards the middle. Ryan Verbstisky explains: “They explore more. They’re not so anxious, swimming around the outside, swimming a million miles an hour. They’re a little bit more chilled out, so they go into the centre, they swim slowly, they go everywhere. They spend more time in different zones. And that’s tracked by that software.”

The more anxious a zebrafish was, the more it stayed to the walls of the tank. The more relaxed a zebrafish was, the more it drifted to the middle of the tank.

“It’s not a perfect measure… I can’t ask this fish if it’s stressed,” Stewart says. But tracking the speed of a fish swimming the edges of its tank is what many researchers call anxiety-like behaviour.

Why zebrafish? The fish are becoming the third most popular animal for medically related science questions, following rats and mice because of their practicality. They are easy to house, easy to feed, and easy to maintain. Andréa Johnson, an undergraduate with two published papers on terpenes and one more about to be published, explains the fish are, “great for studying anxiety, and also memory, conditioned place preference, withdrawal, pharmacology . . .(it’s) really easy to quantify their behaviour.”

These terpenes were chosen because they are in the super-class terpenes. “There are about eight that are considered ‘super-class’ terpenes,” Johnson explains. “ They’re the most common ones that are like ‘this one will give you an uplifting experience. . .’ And so to narrow my focus, I just chose the most common terpenes that are in the (research) discourse but also in consumer discourse as well.”

Now that the research is beginning to show the effects of terpenes, the next step in research is how to block the compound. “People talk about how lavender and stuff can be a relaxing thing — but what is actually happening? And how is that actually working in our brains?” Stewart asks.

The next stage of testing requires researchers to find out which receptors the terpenes are affecting. Verbitsky is one of the researchers who is beginning to look at how to block the receptors that take the terpenes. “So basically at those receptors, it would block the (terpene) from working. So they would act similarly to the control.” He is currently conducting research on the effects of terpinolene on zebrafish.

By blocking the terpenes’ effects on fish through different types of antagonists, this allows the researchers to identify which part of the brain the terpenes are affecting. Right now, the researchers are looking to block cannabinoid receptors CB1 and CB2. CB1 receptors are associated with the psychedelic effects we typically associate with cannabis, while CB2 receptors have shown in previous studies that they actually decrease addictive behaviours in studies with mice.

“The potential here is that we could have a naturally derived anti-anxiety compound without the risk of side-effects or addictive effects. So if it were to go to clinical studies, it could be a new alternative to anxiety medications,” Johnson says. More students, like James Hudson and Ryan Verbistky, are finding mixed results with different types of terpenes. Hudson studied the terpene bisoprolol last semester and found very little change in behaviour, while the research team who published their paper on alpha-pinene found anti-anxiety effects when the terpene was separated.

“I think it’s really interesting, because a lot of the research on cannabis has focused on, probably the things you’ve heard about, like THC and CBD. And there’s lots of other things such as terpenes in cannabis that potentially affect CBD and THC and those effects as well as have their own effects,” says Hudson.

Thanks to the new labs that opened last year, more MacEwan students are furthering the research of terpene effects. While the testing of terpenes is still in
its humble beginnings, the researchers are optimistic about cannabis terpenes having a future in therapeutic practices and for reducing anxiety.

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