Motor neuron development in zebrafish is altered by brief (5-hr) exposures to THC (∆9-tetrahydrocannabinol) or CBD (cannabidiol) during gastrulation

Abstract

Marijuana is one of the most commonly used illicit recreational drugs and is widely used for medicinal purposes. The psychoactive ingredient in marijuana is ∆⁹-tetrahydrocannabinol (∆⁹-THC), whereas the major non-psychoactive ingredient is cannabidiol (CBD). Here, we exposed zebrafish embryos to ∆⁹-THC or CBD for 5 hours during the critical stage of development known as gastrulation. Embryos were allowed to develop normally and were examined at 2 and 5 days post fertilization. THC and CBD treated embryos exhibited reduced heart rates, axial malformations and shorter trunks. Cannabinoid treatment altered synaptic activity at neuromuscular junctions (NMJs), and fluorescent labelling of primary and secondary motor neurons indicated a change in branching patterns and a reduction in the number of axonal branches in the trunk musculature. Furthermore, there were alterations in the α-bungarotoxin labelling of nicotinic acetylcholine receptors at NMJs. Locomotion studies show that larvae exposed to THC or CBD during gastrulation exhibited drastic reductions in the number of C-start escape responses to sound stimuli, but not to touch stimuli. Together these findings indicate that zebrafish embryos exposed to ∆⁹-THC or CBD during the brief but critical period of gastrulation exhibited alterations in heart rate, motor neuronal morphology, synaptic activity at the NMJ and locomotor responses to sound.

Figure 1

Effect of THC and CBD exposure on zebrafish embryos. (A) A schematic of the exposure paradigm of cannabinoids in this study. Red bar shows the duration of the cannabinoid exposure which occurred for 5 hours during gastrulation. (B,C) Embryos were untreated (control), or exposed to 2 mg/L, 4 mg/L, 6 mg/L, 8 mg/L or 10 mg/L THC or 1 mg/L, 2 mg/L, 3 mg/L or 4 mg/L CBD (from 5.25 hpf to 10.75 hpf and then allowed to develop in normal embryo media. Images were taken at 48–52 hpf. (D) Bar graph showing the body lengths of fish in untreated control (black, n = 59), different concentrations of THC (pink, n = 54, 48, 61, 57 and 55 for 2, 4, 6, 8 and 10 mg/L THC-treated fish respectively) or corresponding vehicle control (blue, n = 39, 37, 22, 25 and 20 for 0.2, 0.4, 0.6, 0.8 and 1 percent methanol-treated fish respectively). (E) Bar graph showing the body lengths of fish in untreated control (black, n = 51), different concentrations of CBD (pink, n = 52, 52, 25 and 19 for 1, 2, 3 and 4 mg/L CBD-treated fish respectively) or corresponding vehicle control (blue, n = 36, 39, 32 and 37for 0.1, 0.2, 0.3, and 0.4 percent methanol-treated fish respectively). ^***Significantly different from vehicle control, p < 0.001.

Figure 2

Effect of THC and CBD exposure on survival and hatching. (A,B) Line graph showing the percentage of embryos that survived and hatched within the first 5 days of development following THC exposure during gastrulation (N = 4 experiments and n = 20 embryos for each treatment). (C,D) Line graph showing the percentage of embryos that survived (N = 5 experiment and n = 25 embryos for each treatment) and hatched within the first 5 days after egg fertilization following CBD exposure (N = 3 experiment and n = 25 embryos for each treatment). ^**Significantly different from vehicle control, p < 0.01. ^***Significantly different from vehicle control, p < 0.001.

Figure 3

Effect of THC exposure on heart rate. (A) Bar graph showing the heart rate of untreated control embryos (n = 29), 2–10 mg/L THC-exposed embryos (n = 26, 22, 22, 21 and 21 for 2, 4, 6, 8, and 10 mg/L THC respectively) and corresponding vehicle controls (methanol-treated embryos; n = 31, 25, 35, 26 and 28 for 0.2, 0.4, 0.6, 0.8, and 1 percent methanol respectively). (B) Bar graph showing the heart rate of untreated control embryos (n = 22), 1–4 mg/L CBD-exposed embryos (n = 42, 40, 25 and 20 for 1, 2, 3 and 4 mg/L CBD respectively) and corresponding vehicle treated embryos (n = 25, 31, 24 and 25 for 0.1, 0.2, 0.3 and 0.4 percent methanol respectively). ^***Significantly different from vehicle control, p < 0.001.

Figure 4

Miniature endplate currents (mEPCs) recorded from zebrafish white muscle fibers of vehicle control and 6 mg/L THC-treated embryos (left column), and vehicle control and 3 mg/L CBD-treated embryos (right column). (A) Raw traces obtained from 2 dpf vehicle control (0.6% Methanol, 0.3% Methanol), 6 mg/L THC-treated embryos and 3 mg/L CBD treated embryos. (B) Averaged mEPCs obtained from white muscle (black line) fit with a single exponential decay over the fast component (τ_fast, red dashed line) or slow component (τ_slow, blue dashed line). Averaged mEPCs acquired from vehicle control (0.6% Methanol 6 events; 0.3% Methanol 36 events), 6 mg/L THC-treated embryos (11 events) and 3 mg/L CBD (6 events) (C) Bar graph of the mean mEPC frequency of vehicle and THC-treated embryos (left), and vehicle and CBD-treated embryos (right). ^*Significantly different from vehicle control, p < 0.05. ^***Significantly different from vehicle control, p < 0.001.

Figure 5

Antibody labelling (anti-znp1) of axonal branches of primary motor neurons in 2 dpf embryos in vehicle controls, 6 mg/L THC-treated embryos and 3 mg/L CBD treated embryos. (A–D) Branching patterns and labelling of axons appear to be similar between controls and THC-treated embryos but reduced in CBD treated embryos. (E) Bar graph showing the number of branches emanating from primary motor axons in vehicle control (n = 7) and 6 mg/L THC treated embryos (n = 8), counted from 9 different square areas (each 1500 μm² area). (F) Bar graph showing the number of branches emanating from primary motor axons in vehicle control (n = 6) and 3 mg/L CBD treated embryos (n = 8), counted from 9 different square areas (each about 1500 μm² area). ^*Significantly different from vehicle control, p < 0.01.

Figure 6

Antibody labelling (anti-zn8) of axonal branches of secondary motor neurons in 2 dpf embryos in vehicle control, 6 mg/L THC-treated embryos and 3 mg/L CBD treated embryos. (A–D) Dorsal, ventral and lateral branches emanating from secondary motor neurons are indicated by yellow, white and blue arrows. Dorsal branches were absent in THC and CBD treated embryos (B,D). Fewer lateral branches are visible in CBD treated embryos. (E–G) Bar graph comparing percentage of dorsal branches (E), ventral branches (F) and lateral branches (G) emanating from secondary motor neurons in vehicle control (n = 11) and 6 mg/L THC treated embryos (n = 11). (H–J) Bar graph comparing percentage of dorsal branches (H), ventral branches (I) and lateral branches (J) emanating from secondary motor neurons in vehicle control (n = 11) and 3 mg/L CBD treated embryos (n = 9). ^**Significantly different from vehicle control, p < 0.01. ^***Significantly different from vehicle control, p < 0.001.

Figure 7

Expression of nicotinic acetylcholine receptors (nAChRs) in 2 dpf embryos of controls, 6 mg/L THC and 3 mg/L CBD-treated fish. (A–D) α-bungarotoxin labelling of postsynaptic membranes at the NMJ in zebrafish trunk musculature. (E) Bar graph representing the total number of α-bungarotoxin puncta counted over per 1000 μm² area and compared between vehicle control (n = 10) and 6 mg/L THC-treated embryos (n = 6). (F) Bar graph representing the number of α-bungarotoxin puncta with a minimum area of ~5 μm², compared between vehicle control (n = 10) and 6 mg/L THC-treated embryos (n = 6). (G) Bar graph representing the total number of α-bungarotoxin puncta counted over a 1000 μm² area and compared between vehicle control (n = 8) and 3 mg/L CBD-treated embryos (n = 7). (H) Bar graph representing the number of α-bungarotoxin puncta with a minimum area of ~5 μm², compared between vehicle control (n = 8) and 3 mg/L CBD-treated embryos (n = 7). * Significantly different from vehicle controls, p < 0.05. **Significantly different from vehicle controls, p < 0.01.

Figure 8

Quantification of the response rate of 5-dpf zebrafish larvae to touch and sound stimuli. (A) Bar graph comparing the ratio of larvae responding to a touch stimulus in vehicle control (n = 39 embryos in 4 experiments) and 6 mg/L THC (n = 51 embryos in 4 experiments). (B) Bar graph comparing the ratio of larvae responding to a touch stimulus in vehicle control (n = 42 embryos in 4 experiments) and 3 mg/L CBD (n = 25 embryos in 3 experiments) (C) Bar graph comparing the ratio of larvae responding to a sound stimulus in vehicle control (n = 30 embryos in 5 experiments) and 6 mg/L THC (n = 30 embryos in 5 experiments). (D) Bar graph comparing the ratio of larvae responding to a sound stimulus in vehicle control (n = 24 embryos in 4 experiments) and 3 mg/L CBD (n = 30 embryos in 5 experiments). ^*Significantly different from controls p < 0.05. ^***Significantly different from controls p < 0.001.