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Early Exposure to THC Alters M-Cell Development in Zebrafish Embryos

Cannabis is one of the most commonly used illicit recreational drugs that is often taken for medicinal purposes. The psychoactive ingredient in cannabis is Δ⁹-Tetrahydrocannabinol (Δ⁹-THC, hereafter referred to as THC), which is an agonist at the endocannabinoid receptors CB(1)R and CB(2)R. Here, we exposed zebrafish embryos to THC during the gastrulation phase to determine the long-term effects during development. We specifically focused on reticulospinal neurons known as the Mauthner cells…

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. 2020 Jan 4;8(1):5.

doi: 10.3390/biomedicines8010005.

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Md Ruhul Amin et al. Biomedicines. .

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Abstract

Cannabis is one of the most commonly used illicit recreational drugs that is often taken for medicinal purposes. The psychoactive ingredient in cannabis is Δ9-Tetrahydrocannabinol (Δ9-THC, hereafter referred to as THC), which is an agonist at the endocannabinoid receptors CB1R and CB2R. Here, we exposed zebrafish embryos to THC during the gastrulation phase to determine the long-term effects during development. We specifically focused on reticulospinal neurons known as the Mauthner cells (M-cell) that are involved in escape response movements. The M- cells are born during gastrulation, thus allowing us to examine neuronal morphology of neurons born during the time of exposure. After the exposure, embryos were allowed to develop normally and were examined at two days post-fertilization for M-cell morphology and escape responses. THC treated embryos exhibited subtle alterations in M-cell axon diameter and small changes in escape response dynamics to touch. Because escape responses were altered, we also examined muscle fiber development. The fluorescent labelling of red and white muscle fibers showed that while muscles were largely intact, the fibers were slightly disorganized with subtle but significant changes in the pattern of expression of nicotinic acetylcholine receptors. However, there were no overt changes in the expression of nicotinic receptor subunit mRNA ascertained by qPCR. Embryos were allowed to further develop until 5 dpf, when they were examined for overall levels of movement. Animals exposed to THC during gastrulation exhibited reduced activity compared with vehicle controls. Together, these findings indicate that zebrafish exposed to THC during the gastrula phase exhibit small changes in neuronal and muscle morphology that may impact behavior and locomotion.

Keywords: CNS; Mauthner; NMJ; cannabinoids; motor neurons; muscle.

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1

Figure 1

Δ9-Tetrahydrocannabinol (THC) exposure reduces M-cell axonal diameter. (A,G) Immunolabeling of M-cells with anti-3A10 and anti-RMO44 in a vehicle-treated embryo; (B,H) Higher magnification of M-cell body and axon of vehicle-treated embryos. White arrow shows the cell body of the M-cell. (C,I) Bar graph of the width of an M-cell body in vehicle and THC treated embryos. (D,J) Immunolabeling of M-cells with anti-3A10 and anti-RMO44 in a THC-treated (6 mg/L) embryo; (E,K) Higher magnification of M-cell body and axon of a THC-treated embryo. Red arrow points to the proximal axon immediately anterior to the decussation point. (F,L) Bar graph of the diameter of M-cell axons slightly anterior to the decussation point in vehicle and THC-treated embryos. ** Significantly different from vehicle control, p < 0.005. *** significantly different from vehicle control, p < 0.001.

Figure 2

Figure 2

Exposure to THC during gastrulation alters escape response parameters. Analysis and quantification of C-bend parameters was carried out at 2 dpf. Zebrafish embryos exhibit a C-bend in response to a jet of water directed at the head just behind the eyes. (A) Bar graph shows the maximum angle of bend for vehicle and THC-treated (6 mg/L) embryos. (B) Shows the instantaneous peak speed (mm/s) during c-bend. (C) Shows the instantaneous peak acceleration during C-bend. (D) Bar graph showing the time for the tail to bend to the maximum angle. * Significantly different from vehicle control, p < 0.05.

Figure 3

Figure 3

Co-labeling of red muscle fibers and nAChRs using anti-F59 and Alexa 488 conjugated α-bungarotoxin respectively. (A) α-Bungarotoxin labelled nAChRs associated with red muscle fibers in vehicle-treated embryos. (B) Anti-F-59 labelled muscle fibers. Red arrows point to the edge of a muscle fiber. Inset shows muscle fibers at higher magnification to better determine the size of the fiber. (C) Merged image showing the co-labeled red muscle fiber and nAChR in vehicle-treated animals. (D) α-bungarotoxin labelled nAChRs associated with red muscle fibers in THC-treated (6 mg/L) embryos. White arrow shows the cluster of nAChRs. (E) Anti-F59 labelled muscle fibers. Red arrows point to the edge of a muscle fiber. Inset shows muscle fibers at higher magnification to better determine the size of the fiber. (F) Merged image showing the co-labeled red muscle fiber and nAChR in THC-treated animals. (G) Bar graph showing the diameter of red fibers for vehicle and THC treated embryos and (H) Measurement of red fiber length. *** significantly different from vehicle control, p < 0.001.

Figure 4

Figure 4

Co-labeling of white muscle fibers and nAChRs using anti-F310 and Alexa 488 conjugated α-bungarotoxin respectively. (A) α-Bungarotoxin labelled nAChRs associated with white muscle fibers in vehicle-treated embryos. White arrow shows clusters of nAChRs. (B) Anti-F-59 labelled muscle fibers. Red arrows point to the edge of a muscle fiber. Inset shows muscle fibers at higher magnification to better determine the size of the fiber. (C) Merged image showing the co-labeled white muscle fiber and nAChR in vehicle-treated animals. (D) α-bungarotoxin labelled nAChRs associated with white muscle fibers in THC-treated (6 mg/L) embryos. White arrow shows clusters of nAChRs. (E) Anti-F310 labelled muscle fibers. Red arrows point to the edge of a muscle fiber. Inset shows muscle fibers at higher magnification to better determine the size of the fiber. (F) Merged image showing the co-labeled white muscle fiber and nAChR in THC-treated animals. (G) Bar graph showing the diameter of white fibers for vehicle and THC treated embryos and (H) Measurement of white fiber length. *** significantly different from vehicle control, p < 0.001.

Figure 5

Figure 5

The relative levels of nAChR subunits (α1, γ and ε) mRNAs were analyzed by real-time qPCR. The relative expression was measured from vehicle control and THC treated embryos using expression in vehicle control as calibrator. (A) The relative level of α1 nAChR expression from vehicle and THC treated embryos. (B,C) The relative expression of γ and ε, respectively. Data are expressed as the mean ± SE for individual groups (n = 5).

Figure 6

Figure 6

THC exposure affects free swimming activity (locomotion) of zebrafish embryos at 5 dpf. Bar graphs display changes in larval mean distance moved (A), mean velocity (in mm/s for one hour) (B), mean activity (% rate for one hour) (C), and frequency of swim bouts within one hour (D). *** significantly different from vehicle control, p < 0.001.

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References

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Heartland

Cbd long term effects study by canopy, lifespan increased?

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Visit our community site for vetted suppliers at http://theCBD.place. It’s time that this subject was given more internet exposure. We are here to discuss topics related to medical marijuana and our experiences using CBD. Please do not assume that anyone here is a medical professional.

Source: https://www.reddit.com/r/CBD/comments/jzwr6o/cbd_long_term_effects_study_by_canopy_lifespan/

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CBD firm on high growth track surpasses $5M in sales

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TAMPA — Proleve Distribution, a Tampa based manufacturer of all-natural CBD products, plans to hire 20 people to support a period of hyper-growth.  

Founded in 2017, Proleve, which produces and distributes products containing the hemp extract CBD, or Cannabidiol, will close out 2020 at $5 million in annual revenue, according to a statement. That’s up 178% from $1.8 million in 2019. To keep up with growing demand, Proleve is now in the early stages of building a new distribution center near Tampa International Airport, the release adds. New jobs added in the coming weeks will help support operations at the facility, as well as at the company’s existing South Tampa headquarters, which will be converted into a retail store. 

“In a year that’s been marked with challenges for so many companies and industries, we’re pleased that Proleve has not only been able to withstand such challenges, but overall has actually been able to thrive in this environment,” Proleve Founder and President Jonathan Solomon says in the statement. “With that in mind, we’ve been working really hard to seize this opportunity in order to further position our growth.”

Earlier this year, Proleve became among the first CBD companies to be officially licensed and certified by Florida’s Department of Agriculture and Consumer Services under new regulations. The certification makes Proleve an official state-approved source for food products containing CBD, in addition to being FDA-registered, the release states.

“Having this official status within the state of Florida has meant an added sense of security for our customers,” Solomon says. “It’s turned out to be an important piece of the puzzle this year, even more so than expected, with sales jumping as our products have offered a sense of relief for those who have faced hardship in 2020.”

“During the peak of COVID-19 earlier this year, we were more focused on keeping jobs safe,” Solomon adds. “Now that we see the full extent of how our business plays into the economic recovery of this region, we’re glad to be adding more jobs and serving as a catalyst for growth.”

Proleve’s products are sold in stores nationwide and worldwide. Its products, including tincture, topicals, capsules and bath bombs are all vegan, kosher-certified, third-party lab tested and 100% natural.

                                                                         

Source: https://www.google.com/url?rct=j&sa=t&url=https://www.businessobserverfl.com/article/cbd-firm-on-high-growth-track-surpasses-dollar5m-in-sales&ct=ga&cd=CAIyGjQ2MzM1ZTBkNWQyZmIyYzc6Y29tOmVuOlVT&usg=AFQjCNESyacJcEc1fMFdEgw7Um8nuECjbA

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Home Grown: Community reports minty and floral flavors from hemp green

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CBS 13’s April Hettinger samples Yuma’s newest leafy crop

YUMA, Ariz. (KYMA, KECY) – Hemp leafy greens were put to the test to see if their flavors are worthy enough to make it in bagged mixes in the grocery stores.

81 people took a survey of nine samples the past two Fridays.

Staff was able to narrow down the 17 types of hemp to five that were the best tasting.

The community sampled the five hemp greens along with three commonly available greens like romaine, kale and baby leaf spinach for comparison.

The ninth sample was a pesto made from the hemp greens by local chefs.

The grower, Robert Masson, agriculture extension agent with the University of Arizona’s Yuma County extension explains the overall consensus of this unique lettuce.

“In general, the hemp was really well liked. Some people didn’t like it, but most of the people did,” Masson said. “They reported minty flavors, fruity flavors and just a little bit of floral.”

Attendees rated the crispiness of the hemp to be similar to kale.

It also has more peach fuzz than other greens.

In its overall likeability, the community rated romaine and spinach higher than the hemp at a 4.1 on a one through five scale.

Hemp was rated a 3.3.

The best tasting hemp greens might make it into grocery stores as bagged mixes for health benefits.

Source: https://www.google.com/url?rct=j&sa=t&url=https://kyma.com/news/home-grown/2020/11/23/home-grown-community-reports-minty-and-floral-flavors-from-hemp-green/&ct=ga&cd=CAIyGjYxZmRhODRmNjY4MjhhNTU6Y29tOmVuOlVT&usg=AFQjCNE7rcoLiE_6doYx8sPtN8OaaibwHQ

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