In zebrafish embryos, chronic or acute exposure to (synthetic CB1 agonist) ACEA resulted in morphological symptoms of Fetal Alcohol Spectrum Disorder (FASD), similar to chronic high alcohol exposure. This effect was rescued by CB1 antagonist SR141716A. Dual inhibition of endocannabinoid degrading enzymes FAAH and MAGL (JZL195) together with sub-threshold alcohol produced similar effects. Finally, while neither ACEA not alcohol alone affected behavior, in combination they increased risk taking behavior (Boa-Amponsem et al., 2019). These results suggest that endocannabinoids are involved in normal fetal development, alcohol can induce FASD via CB1 and a combination of cannabis and alcohol use during pregnancy may increase risk of FASD.
In zebrafish embryos it was found that CB2 regulates embryonic hematopoietic stem cell development with CB2 activation stimulating stem cell proliferation and CB2 inhibition blocking stem cell proliferation (Esain et al., 2015).
In chicken embryos, the synthetic cannabinoid HU-210 reduced viability by 100% at 10 M and CBD reduced viability by 80% at 50 M (Gustafsson and Jacobsson, 2019). Although this suggests cannabinoids are embryotoxic please note that chickens are genetically quite different from humans and the doses used here are very high for human standards.
Cultured chicken embryonic retinal glial progenitor cells express CB1 and CB2 and their stimulation with synthetic cannabinoid WIN55,212-2 (0.5 – 5.0 M) reduced progenitor proliferation, increased mitochondrial oxide radical formation and primed cells for calcium signaling. Similar results were obtained after MAGL and FAAH treatment suggesting a role for the ECS in retinal development and cell differentiation (Freitas et al., 2019).
Mice deficient for CB1 and/or CB2 have longer femur bones than wildtype mice. Also, THC slows skeletal growth in wildtype and CB2 deficient, but not CB1 deficient mice, resulting in lower body weight (Wasserman et al., 2015). The results imply CB1 in bone physiology and embryonic growth and may explain the tendency for reduced birth weight in mothers who use cannabis during pregnancy.
In mice, CB1 signaling is operational in subcortical proliferative zones from embryonic day 12 in the telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells. When layer patterning is accomplished, developing pyramidal cells rely on CB1 signaling to initiate the elongation and fasciculation of their long-range axons (Mulder et al., 2008).
In mice, CB1 is enriched in the axonal growth cones of cortical GABAergic interneurons during late gestation. endocannabinoids trigger CB1 internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA (Berghuis et al., 2007) suggesting a regulatory role for CB1 in axonal pathfinding and synaptogenesis.
In the offspring of baboons subjected to maternal nutrient reduction, male but not female fetuses showed reduced CB1 expression in the temporal cortex (Gandhi et al., 2019) suggesting fetal ECS is sensitive to environmental changes.
In mouse fetuses and human pancreatic islets, α cells produce 2-AG, which primes the recruitment of β cells by CB1 activation. AEA impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of TRPV1 and CB1. TRPV1 deficiency increases islet size whereas CB1R deficiency augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1 deficient islet microstructure and improves coordinated hormone secretion (Malenczyk et al., 2015).
In zebrafish the effect of THC (0.3, 0.6, 1.25, 2.5, 5 mg/L (1, 2, 4, 8, 16 μM)) and CBD (0.07, 0.1, 0.3, 0.6, 1.25 mg/L (0.25, 0.5, 1, 2, 4 μM) on gestation was followed from blastula to larval stages. Despite the similarity in THC and CBD dysmorphologies, i.e., edemas, curved axis, eye/snout/jaw/trunk/fin deformities, swim bladder distention, and behavioral abnormalities, the LC50 for CBD (0.53 mg/L) was nearly seven times lower than THC (3.65 mg/L). Also CBD was more bioconcentrated compared to THC despite higher THC water concentrations (Carty et al., 2017). This suggests that chronic cannabinoid exposure during gestation can lead to persistent developmental abnormalities but please note that zebrafish are genetically quite different from humans and the doses used here are very high for human standards.
Zebrafish embryos exposed to THC (6 mg/L) during the gastrula phase exhibit small changes in neuronal and muscle morphology that may impact behavior and locomotion (Amin et al., 2020).
Another zebrafish experiment found that incubation in 20-300 μg/L CBD slightly delayed hatching and transiently increased embryonic motor activity but did not induce teratogenicity or neurotoxicity (Valim Brigante et al., 2018).
Zebrafish embryos exposed to varying concentrations of CBD (0.02, 0.1, 0.5 μM) during larval development and assessed aging in both the F0 (exposed generation) and their F1 offspring 30 months later. F0 exposure to CBD significantly increased survival (~ 20%) and reduced size (wet weight and length) of female fish. While survival was increased, the age-related loss of locomotor function was unaffected and the effects on fecundity varied by sex and dose. Treatment with 0.5 μM CBD significantly reduced sperm concentration in males, but 0.1 μM increased egg production in females. Similar to other model systems, control aged zebrafish exhibited increased kyphosis as well as increased expression markers of senescence, and inflammation (p16ink4ab, tnfα, il1b, il6, and pparγ) in the liver. Exposure to CBD significantly reduced the expression of several of these genes in a dose-dependent manner relative to the age-matched controls. The effects of CBD on size, gene expression, and reproduction were not reproduced in the F1 generation, suggesting the influence on aging was not cross-generational (Pandelides et al., 2020a).
In another experiment by the same group zebrafish were exposed to different concentrations of THC (0.08, 0.4, 2 μM) during embryonic-larval development and the effects on aging were measured 30 months later and in the offspring of the exposed fish (F1 generation). Exposure to 0.08 μM THC resulted in increased male survival at 30 months of age. As the concentration of THC increased, this protective effect was lost. Treatment with the lowest concentration of THC also significantly increased egg production, while higher concentrations resulted in impaired fecundity. Treatment with the lowest dose of THC significantly reduced wet weight, the incidence of kyphosis, and the expression of several senescence and inflammatory markers (p16ink4ab, tnfα, il-1β, il-6, PPARα and pparγ) in the liver, but not at higher doses indicating a biphasic or hormetic effect. Exposure to THC did not affect the age-related reductions in locomotor behavior. Within the F1 generation, many of these changes were not observed. However, the reduction in fecundity due to THC exposure was worse in the F1 generation because offspring whose parents received high dose of THC were completely unable to reproduce (Pandelides et al., 2020b). Interestingly the same group did observe some transgenerational effects of CBD and THC such as dazl expression and photomotor activity (Carty et al., 2018), indicating that the used dose of cannabinoid and the choice of parameter determine whether embryonic exposure has positive or negative consequences and whether or not these effects are transgenerational.
In another zebrafish experiment, cannabinoids like THC, CBD, HU-210 or CP-55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, cannabinoids reduced Shh signaling via CB1 (Fish et al., 2019).
Zebrafish embryos exposed to ∆9-THC (2-10 mg/l) or CBD (1-4 mg/l) during the brief but critical 5-hour period of gastrulation exhibited alterations in heart rate, motor neuronal morphology, synaptic activity at the NMJ and locomotor responses to sound (Ahmed et al., 2018).
Injection of THC (3 mg/kg i.p.) in pregnant mice (embryonic day 12-16) interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. Fetal THC also increased seizure susceptibility in offspring suggesting an important role for CB1 in fetal development (de Salas-Quiroga et al., 2015) although it should be noted that the used dose here is very high for human standards.
In cultured human cerebral organoids from human embryonic stem cells reminiscing the developing fetal brain, 1 M THC (added to the growth medium for 3 days) resulted in reduced neuronal maturation, reduced neurite outgrowth, reduced CB1 expression and reduced spontaneous neuronal firing (Ao et al., 2020). Although the results suggest a detrimental effect of THC on brain development it should be noted that the experimental system is far removed from real human brain development and 3-day continuous exposure to 1 M THC is unlikely to be achieved in the developing fetal brain.
In immature cortical neurons derived from human induced pluripotent stem cells, CB1, but not CB2R, GPR55 or TRPV1, is expressed. 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the decrease in neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. The results suggest human stem cell-derived neurons may be a useful system to test the effect of plant cannabinoids on human brain development (Shum et al., 2020).
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