Neurotoxic or Protective Cannabis Components: Delta-9-Tetrahydrocannabinol (<sup>Δ9</sup>THC) and Cannabidiol (CBD)

doi:10.14218/JERP.2023.00017

Review Article

OPEN ACCESS

Download PDF

Neurotoxic or Protective Cannabis Components: Delta-9-Tetrahydrocannabinol (^Δ9THC) and Cannabidiol (CBD)

Marilyn H. Silva^*

Journal of Exploratory Research in Pharmacology 2023;8(4):299-322

doi: 10.14218/JERP.2023.00017

Received: March 6, 2023

Revised: April 15, 2023

Accepted: April 19, 2023

Published online: July 12, 2023

Author information

Citation: Silva MH. Neurotoxic or Protective Cannabis Components: Delta-9-Tetrahydrocannabinol (^Δ9THC) and Cannabidiol (CBD). J Explor Res Pharmacol. 2023;8(4):299-322. doi: 10.14218/JERP.2023.00017.

Abstract

Cannabis sativa contains phytocannabinoids that are psychoactive and neurotoxic (delta-9-tetrahydrocannabinol: ^Δ9THC) or nonpsychoactive and presumptively neuroprotective (cannabidiol: CBD). Along with rising legalization, availability, and demand, the ^Δ9THC:CBD ratio also has increased. Cannabis legalization means that use will likely increase in pregnant or breastfeeding women, affecting all stages of brain and neurodevelopment of their offspring. ^Δ9THC exposure in utero or during development leads to lasting detrimental effects on behavior, cognition, locomotor activity, as well as epigenetic changes. Caution is urged with cannabis use. CBD is one of the most actively studied therapies for a broad spectrum of neurological, inflammatory, and mental diseases (e.g., Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, schizophrenia) because of its efficacy, low toxicity, and availability. While data indicate that the benefits of CBD may outweigh its risks, there are indications that it poses a risk for adverse effects on neurodevelopment from in-utero exposure as well as detrimental effects on male reproduction. Therefore, there is a clear need to continue researching the effects of ^Δ9THC exposure as well as the optimal CBD treatment related to disease management while stressing the need to further characterize possible adverse effects.

Keywords

^Δ9THC, Delta-9-Tetrahydrocannabinol, Cannabidiol, Neurodevelopment, Endocannabinoid system, Cannabis, Neuroprotection, Neurotoxicity

Introduction

Medicinal and recreational cannabis use has increased globally, and continuation of this trend is anticipated as its use becomes legalized internationally.^1,2Cannabis sativa is composed of over 100 “cannabinoids,”^3,4 but the psychoactive compound delta-9-tetrahydrocannabinol (^Δ9-THC), isolated in 1964, and the nonpsychoactive compound cannabidiol (CBD), isolated in 1940,⁵ represent the most abundant components. Consumption of cannabis products occurs through diverse routes (inhaled smoke, vaping of liquid extracts, resins or waxes, lotions, edibles).^6,7 Inhaled cannabinoids are rapidly absorbed in the lungs⁸ but less so by other routes (e.g., dermal, oral, rectal).⁹ Due to their highly lipophilic properties, they are stored in adipose tissue for weeks or months and are concentrated in the breast milk of rodents and humans.^10,11 CBD products can have beneficial health effects and aid in various medical disorders (e.g., Parkinson’s disease, anxiety, and epilepsy).^12,13 Accumulating evidence also indicates there are neurotoxic and reproductive effects from exposure.^14–18

Due to increasing cannabis use, exposure to ^Δ9-THC presents concerning health risks because use will likely also increase in pregnant or breastfeeding women, affecting all stages of brain and neurodevelopment of their offspring.^19–24 Along with increased legalization, social acceptance, and use, a change in the ratio of^Δ9-THC to CBD in cannabis has also occurred, leading to a change in potency (the ^Δ9-THC:CBD ratio increased from 14:1 in 1995 to 80:1 in 2014).²⁵ Ultimately, the extent of cannabis neurotoxicity²⁶ is dependent on many variables, including the ^Δ9-THC exposure level, purity,²⁵ route of administration,^7,9,27 developmental age at exposure,^23,28–30 health status,^31,32 pregnancy status,^21,33–36 lactational status,^37,38 and others.³⁹ Further, due to the lipophilic nature of these compounds, it has been shown that exposure at low, realistically achievable in-vivo concentrations causes specific molecular targets to be affected, resulting in behavioral or cognitive deficits in those with ^Δ9THC exposure,^39–41 or potential benefits that greatly improve the health of those with neurodegenerative diseases.^42–44

In this review, both the risks and benefits of exposure associated with ^Δ9THC and CBD were investigated. Notably, the risks from CBD exposure, which is usually considered to be safe, are associated with reproductive and developmental health effects.⁴⁵ Recently, concerns have been raised about CBD use, since it is available in numerous over-the-counter products, with little data supporting its safety or efficacy.⁴⁶ The side effects and adverse health effects, along with questions regarding the ingredients, are often unknown. On the other hand, ^Δ9THC exposure has been associated with adverse effects, depending on the dose, yet the benefits of this drug need to be emphasized. These phytocannabinoids were selected because they are the dominant compounds in cannabis, and they are often used as treatments for physical ailments as well as for recreational use. There is a vast amount of literature characterizing these compounds and their effects during development and throughout life in both animal and human studies, but it is important to present the risks as well as the benefits.

The endocannabinoid system (eCBS)

The eCBS was discovered in the 1990s while investigating the mode of action (MOA) of ^Δ9THC. It is innate and multifaceted, affecting metabolic pathways throughout the body [e.g., muscle, adipose tissue, gastrointestinal tract, liver, and central nervous system (CNS)].⁴⁷ It helps to shape neuronal connectivity in the brain throughout development and into adulthood,⁴⁸ affecting the gamma-aminobutyric acid (GABA)ergic, glutamatergic, opioid, and dopaminergic systems.⁴⁹ Cell membrane-bound cannabinoid-1 receptors (CB1Rs) are the most abundant in the brain, while CB2Rs are mainly expressed on immune cells (T-cells, macrophages) in the periphery or glia/microglia in the brain.^47,50 Some researchers have suggested that the transient receptor potential cation channel subfamily V member 1 (TRPV1 or vanilloid receptor 1) could be classified as CB3R, as it is activated by CBD.⁵¹ Each receptor type can act independently; however, depending on their location, CB1Rs and CB2Rs (possibly also CB3Rs) can act together, competitively, or in opposite directions, potentially through dimerization to regulate physiological effects.

Normally, neurotransmitters [e.g., glutamate, GABA, serotonin (5-HT), dopamine (DA), acetylcholine (ACh), or norepinephrine] in the CNS are released presynaptically via neuronal stimulation, or by G protein-coupled receptors and voltage-gated ion channel calcium (Ca⁺²) and potassium (K⁺) influx.^50,52 However, the elevation in postsynaptic Ca⁺² affected by neurotransmitters/receptors through the ion channels [e.g., ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA), or GABA],⁵³ stimulates endocannabinoid (eCB) postsynaptic biosynthesis.^50,52,54

There are two principal eCB ligands [2-arachidonoylglycerol (2-AG) and anandamide (AEA)], which are synthesized postsynaptically from arachidonic acid by N-acyl phosphatidylethanolamine phospholipase D and diacylglycerol lipase alpha/beta (DAGLα/β), respectively.^55–57 These eCBs are produced, as needed,⁴⁷ postsynaptically by Ca⁺²-dependent transacyclase and other enzymes, then they migrate from postsynaptic neurons to the presynaptic CBR.^53,58 Signaling then occurs as CBR couples to the guanosine-5′-triphosphate (G_i/o)/α-protein subunit dimer^58,59 and binds adenyl cyclase to generate cyclic adenosine monophosphate. The cascade decreases presynaptic Ca⁺² influx by blocking the activity of voltage-dependent N-, P/Q- and L-type Ca⁺² channels^60,61 and activation of some K⁺ channels.^53,62 The retrograde eCB (AEA and 2-AG) transmitters in the brain presynaptically inhibit the release of the neurotransmitters GABA,^63,64 glutamate,^63,65,66 DA,^65,67,68 norepinephrine,⁶⁹ 5-HT^67,70 and ACh,^71,72 thereby decreasing the probability of neurotransmitter release. eCBs are then degraded by the serine hydrolase monoacylglycerol lipase (MAGL) in the presynaptic cell and fatty acid amide hydrolase (FAAH) located in the postsynaptic cell.^49,57,73

Figure 1 compares the lipophilic structures of the eCBs (2-AG and AEA) with cannabinoids (e.g., ^Δ9THC and CBD). ^Δ9THC and CBD toxicity or neuroprotection depends on factors such as potency, exposure, duration/frequency, vehicle, route of administration, and species-specific differences. Pharmacokinetic and pharmacodynamic parameters determine the extent of P450 (CYP1A, 3A4, 2C9, and 2C19) metabolic activation and glucuronidation elimination of ^Δ9THC and CBD.^9,74 A tipping point leading to an adverse health effect would depend on an individual’s ability to handle various exposure loads based on age, genetic makeup, health status, and diet, among other influences.^75,76 These risk factors are often difficult to characterize in humans, since hepatic metabolism studies are, by necessity, generally performed in vitro.⁷⁵

Lipophilic structures for delta-9-tetrahydrocannabinol (<sup>Δ9</sup>THC) and cannabidiol (CBD) as well as the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA).

Fig. 1 Lipophilic structures for delta-9-tetrahydrocannabinol (^Δ9THC) and cannabidiol (CBD) as well as the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA).

Each compound acts at the G protein-coupled receptors cannabinoid 1 and 2 receptors, which affect neurotransmitter release.

^Δ9THC-associated mechanisms and neurotoxicity

To understand the effects of ^Δ9THC on the brain, it is helpful to know which areas are affected. The eCBS/CBRs throughout the brain⁷⁷ help to regulate glutamatergic (excitatory), GABAergic (inhibitory),^78,79 dopaminergic, and serotonergic neurotransmitter release at presynaptic terminals.^80,81 The interactions among these systems are complex, occurring via direct and indirect stimulation, which may or may not be overseen by the eCBS to regulate neuroplasticity and excitability toward locomotor activity, cognition (learning and memory), executive functions, reward, motivation, and neuroendocrine control, among other functions.^{78,80,82–86} The striatum in the basal ganglia contains inhibitory GABAergic medium spiny neurons that are affected by the glutamatergic (AMPAR) and dopaminergic (i.e., D1 and D2) receptor inputs from the ventral tegmental area (VTA), substantia nigra (SNc), and prefrontal cortex (PFC).⁸⁷

Table 1 summarizes some of the main brain regions, pathways, and neurotransmitters involving the neuronal connections in the eCBS and affected by ^Δ9THC.^{53,78,80,81,84,85,87–98}

Table 1

Brain regions and pathways affected by endocannabinoids, ^Δ9-THC and/or CBD

Neurotransmitter/Pathway	Brain region associations	Behavior/processes involving eCBS	Reference
Dopamine: DA
Mesolimbic	DA from ventral tegmental area (VTA; midbrain) → ventral striatum (amygdala, pyriform cortex, lateral septal nuclei, nucleus accumbens)	Reward-related cognition (e.g., incentive: wanting; pleasure: liking; positive reinforcement, associative learning) & emotion	⁷⁸^,⁸⁰^,⁸¹^,⁸⁸^–⁹¹
Mesocortical	DA from VTA (midbrain) → prefrontal cortex + hippocampus	Cognition: executive function (e.g., planning, attention, working memory, planning, self-control, etc.), emotion
Nigrostriatal	DA from substantia nigra (pars compacta; substantia nigra SNc: midbrain) → dorsal striatum (i.e., caudate nucleus + putamen)	Neuromotor function, reward-related cognition, associative learning
Tuberoinfundibular	DA from the hypothalamic arcuate (infundibular) + paraventricular nucleus → pituitary gland median eminence	Inhibits the release of prolactin.
Glutamate
Glutamatergic	Hippocampus, neocortex and over 90% of synapses in human brain.	Excitatory effects on VTA & SNc neurons, memory, learning, neural communication	⁵³^,⁹⁰^,⁹²^,⁹³
ɣ-Aminobutyric Acid: GABA
GABAergic	Hippocampus, thalamus, basal ganglia, hypothalamus, brainstem^a	Inhibitory effects on VTA and SNc neurons	⁹⁰^,⁹⁴^–⁹⁶
Serotonin: 5HT
Serotonergic	Dorsal raphe nuclei, cortex, hippocampus	Modulator of receptors with effects depending on subtype (i.e., biphasic effect on VTA neurons)	⁸⁰^,⁸⁴^,⁸⁵^,⁹⁷^,⁹⁸

^aGABAergic transmission includes inhibitory median spiny neurons in the striatum/basal ganglia affected by the glutamatergic (AMPAR) and dopaminergic (D1 and D2) receptor inputs from the VTA, SNc, and PFC.⁸⁷ 5HT, 5-Hydroxytryptamine; AMPAR, Alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor; D1 Receptor, Dopamine receptor subtype 1; D2 Receptor, Dopamine receptor subtype 2; DA, Dopamine; GABA, gamma-aminobutyric acid; Glu, Glutamate; PFC, Prefrontal Cortex; SNc, Substantia Nigra Pars Compacta; SNc, Substantia Nigra Pars Compacta; VTA, Ventral Tegmental Area; VTA, Ventral Tegmental Area.

Cannabinoid signaling can be disrupted through agonistic activity of ^Δ9THC at the CB1Rs throughout areas of the brain. This process leads to inhibition of accumulation of 2-AG and AEA in the brain.^73,99,100 While there are many other neuronal circuits associated with the eCBS, the ones mentioned above are most frequently associated with cannabis.

^Δ9THC-associated neurotoxicity in rodent and nonhuman primate models

^Δ9THC exposure throughout all life stages is associated with effects on behavior, cognition, locomotor activity, birth weight, learning, and other adverse effects.^101–104 Cannabis smoke was listed as a reproductive toxicant on 3 January 2020, under California’s Proposition 65.¹⁰⁴ However, to control for the dose intake and other technical issues, many neurodevelopmental studies performed in animals used intravenous (i.v.) ^Δ9THC administration. Although this is not a likely exposure scenario for humans, the immediate absorption by i.v. could be compared to pulmonary exposure by inhalation.^105,106 Subcutaneous (s.c.), oral (i.e., gavage), and intraperitoneal (i.p.) administration are more slowly absorbed and are subject to local metabolic processes prior to entering the blood stream.^107,108 Other considerations contributing to potential variabilities in evaluating the study results are as follows: 1) often only a single exposure dose was used, limiting potential observations of a dose–response relationship; 2) ^Δ9THC dosing vehicles varied among studies; 3) different species/strains of rodent were used; 4) different exposure scenarios were used; and 5) many different laboratories contributed to the list of studies.

Gestational exposure to ^Δ9THC

The eCBS is involved in the earliest developmental stages, including fertilization, implantation, and neuronal progenitors in the brain, leading to migration, morphogenesis, and axonal guidance.^94,109,110 The effects of ^Δ9THC on these processes can be seen in rodents’ pulmonary exposure by inhalation.^105,106 Administration via a s.c., oral, or i.p. route is more slowly absorbed and is subject to local metabolic processes prior to entering the blood stream.^107,111Δ9THC has profound effects on CB1Rs in areas of the brain regulating GABA, 5-HT, glutamate neurotransmitters, and DA release, influencing, for example, the development of locomotor activity, cognition, learning, memory, and emotional regulation (Table 2).^{11,29,34,112–141} Notably, the lowest doses of ^Δ9THC (0.15 mg/kg/day) in the offspring of Long-Evans rats treated in utero affected preproenkephalin, an endogenous opioid precursor in the nucleus accumbens, amygdala, and striatum, in addition to showing evidence of decreased cognition and other behavioral effects.^112–114 Treatment in utero or from paternal exposure during a full cycle of sperm development, even at low ^Δ9THC doses (0.15 mg/kg/day), resulted in developmental deficits and epigenetic transmission.^{112,113,115–117} Male Wistar adult rats treated throughout sperm development (gavage, 2.0 mg/kg/day) had offspring with affected locomotor activity, feeding behavior, and visual operant signaling.¹¹⁸ Moreover, epidemiological evidence supported findings that cannabis exposure during gestation or during male sperm development results in children with cognitive, motor, and behavioral (including severe psychoses) effects.^33,142–145 Infants with gestational exposure to cannabis may show an exaggerated startle response or an inability to adapt to novel stimuli.^146,147 Furthermore, women who used cannabis during pregnancy had an increase in fetal deaths, premature births, heart rhythm disorders, and fetal intrauterine growth restrictions.³⁶

Table 2

Neurotoxic and behavioral effects from ^Δ9THC treatment during development in animal studies

Animal strain/Sex/Duration/Dose/Vehicle	Day tested	Effects	LOEL (mg/kg/day)	Reference
^Δ9THC in animal studies
Gestational treatment
Long-Evans Dam: GD 5-PND 2; F1 fostered PND 2–21. Dose: i.v. 0.15 mg/kg/day. Vehicle: Tween 80/saline	F1 M/F Pups: PND 2 or PND 62, Adult	NAc: ↓striatal DRD2 mRNA expression; ↓DR2 receptor & binding sites; epigenetic regulation of DRD2 mRNA expression disrupted; affected DA receptor gene regulation. Significance: Increase in sensitivity to opiate reward in adulthood	0.15*	¹¹²
Long-Evans Dam: GD 5-PND 2 fostered PND 2-21. Dose: i.v. 0.15 mg/kg/day. Vehicle: Tween 80/saline	F1 M Pups PND 55, Adult	↓PENK mRNA expression NAc (pup), ↑PENK in NAc & amygdala (adults); ↑Self-administer heroin; ↓latency between active lever press; ↑active lever press; ↑responses on stress test; ↑total responses on active lever on 1st & last extinction days; ↓distance traveled during acquisition & maintenance. Significance: Increased opioid seeking behavior (motivation/reward) & stress response in adulthood	0.15*	¹¹⁴
Long-Evans Dam every 3rd day; PND 28–49; mated PND 64–68; F1 fostered. Dose: i.p. 1.5 mg/kg/day. Vehicle: saline/Tween 80	F1 M/F Pups: PND 35 (Adolescence) or PND 62 Adult	Striatal dysregulation of CB1R gene expression, affecting striatal plasticity; ventral to dorsal striatum disruptions between adolescence & adulthood; F ↓novelty seeking. Significance: Supports relevance to age-dependent vulnerability for neuropsychiatric disorders	1.5*	¹³⁰
Long-Evans Dam every 3rd day PND 28–49; mated PND 64–68; F1 fostered. Dose: i.p. 1.5 mg/kg/day. Vehicle: saline/Tween 80	F1 M/F Pups: PND 35 (Adolescence) or PND 62, Adult	Epigenetic effects & altered CB1R mRNA expressions in NAc associated with glutamatergic system regulation; F ↓ locomotor activity. Significance: Cross-generational epigenetic vulnerability to drug abuse	1.5*	¹¹⁷
Wistar Dam: GD 15–PND 9. Dose: Gavage 3.0 mg/kg/day. Vehicle: sesame oil	F1 M Pup: PND 90, Adult	Disrupted hippocampal GABAergic system; ↓GABA outflow & uptake in hippocampus; ↓ CB1 binding; cognitive impairments. Significance: Long term cognitive deficiency & disrupted GABA neuronal development	5.0*	¹¹⁵
Wistar Dam: GD 5–14, 16, 18, 21 & PND 1 & 5. Dose: Gavage 5.0 mg/kg/day. Vehicle sesame oil	F1 M/F GD 14, 16, 18, 21 + PND 1 & 5 Neonate	Disrupted tyrosine hydroxylase gene activation (rate limiting in DA production); ↑ DOPACL DA metabolite forebrain. Significance: Tyrosine hydroxylase plays a large part in neurodevelopment through DA production	5.0*	¹³¹
Wistar Dam: GD 7–22. Dose: i.p. 3 mg/kg/day. Vehicle: Not stated	F1 M/F Behavior PND 70–100	M: ↓Time on light side of test box (↑anxiety); ↑transition to light; ↓Time in open arm of EPM; ↑VTA spike activity; ↓DA & NMDAR2B PND 21; ↑GAD87 PND 21; F: ↑GAD67, vGLUT1-2; PPARα & PPARϒ1-2 & NMDAR2B in the mesolimbic system (VTA-NAc); M/F: ↑Altered fatty acid concentrations in the nucleus accumbens core & shell up to PND 120 (M) or PND 21 (F). Significance: Sex difference with M more affected than F; Fatty acid deficits disrupt the DA/GLUT/GABAergic neurotransmissions affecting neurodevelopment	3.0*	¹³²
SD Dam: GD 5–PND 2 foster-nursed PND 2–21. Dose: i.v. 0.15 mg/kg/day. Vehicle: Tween80/saline	F1 M/F Pups: PND 22, 45 & 60 Weaning, adolescent, adult	Pup: ↓anxiety: ↓active place avoidance acquisition; ↑active place avoidance reversal phase entries; Adult: ↓attention (acquisition, reversal & distraction) & cognition. Significance: Decreased anxiety, attention & cognitive function	0.15*	¹¹³
SD Dam: Group 1: GD 5–20. Group 2: GD 5–20 + PND 15. Dose: s.c. 2.0 mg/kg/day; PND 15 2.5 mg/kg/day. Vehicle: Tween80/saline	F F1 Pups: Groups 1 & 2: PND 15–28 Juvenile	Group 1 & 2: Male behaviors affected: ↑distance traveled; ↓stretch-attend postures; Group 2: ↓ latency in passive avoidance training; ↑AMPA from DA cells; ↓stretch-attend postures; ↓DA 240 min postacute dose. Significance: Behavioral effects from mesolimbic (NAc) dopaminergic disruptions are greater in males & greater after ^Δ9THC challenge	2.0*	¹³³¹
SD Dam: GD 5–GD 20. Dose: s.c. 2.0 mg/kg/day. Vehicle Tween 80/saline	F1 M/F Pups: Tests done PND 24–28, Juvenile	VTA DA neuron effects: ↑ firing rate; ↓ cells/track; ↓ spikes/burst, burst rate; ↓after hyperpolarization period; ↑DRD2 sensitivity & acute stress vulnerability; ↑ activity, ↓ PPI average in acute restraint & forced swim test. Significance: Sensorimotor gating deficits leading to an increase in susceptivity to stimuli triggering psychotic-like behaviors	2.0*	¹³⁴
SD M Adult 28 days; mated 2 days post dose. Dose: s.c. 2.0, 4.0 mg/kg/day. Vehicle: Tween 80/saline	F1 M Pups: PND 30, 60, 100 & 150 Adolescent, adult	↓ACh activity; ↑ ChAT: ACh biomarker for number of ACh terminals in striatum; ↓ ChAT hippocampus; ↓HC3/ChAT (ACh activity index) n frontal/parietal cortex & striatum. Significance: Paternal ^Δ9THC leads to disruptions in developmental trajectory of ACh potentially affecting attention	2.0	¹¹⁶
Wild-type Mouse Dam: GD 12.5–16.5. Dose: i.p. 3.0 mg/kg/day. Vehicle: saline/DMSO/Tween 80	F1 M/F Pups: PND 20; 2 months; Juvenile, adult	CB1R →affected cortical neuron synaptic signaling development →affected connectivity in cortical GABAergic & glutamatergic systems → ↓fine motor skills; ↓ skilled motor function; 2 months: ↓ success in pellet retrieved in skilled steps test; ↑seizure. Significance: Disrupted CB1 signaling leading to disrupted glutamate & GABA signaling leads to increased susceptibility to seizures and cortico-spinal function.in adulthood	3.0*	¹³⁵
C57Bl/6 Mouse Dam: GD 14.5–18.5. Dose: i.p. 3.0 mg/kg/day. Vehicle: DMSO	F1 M/F: GD 18.5; PND 10 & 120, Fetal, pup, adult	↓ CB1R & misrouted hippocampal CB1R afferents, ↑CB1R density in striatum; Impaired LTD in pyramidal cell synapsis; ↓ synaptic plasticity in the cortical circuitry; Impaired cortical axonal development; ↓2-AG signaling, ↓ CB1R & ↑ MAGL expression, ↓DAGL; abnormal growth cones & cytoskeleton in axonal region. Significance: Abnormal axonal development in growth cone disrupts neuronal circuitry, memory encoding, cognition & executive skills	3.0*	¹³⁶
Postnatal Treatment
Wistar Dam: PND 1–10. Dose: s.c.2.0 mg/kg/day. Vehicle: DMSO/cremophore/saline	F1 M/F Pups: PND 10, 15, 20; 9–21 Preweaning, juvenile	↓Bodyweight gain; GABA excitatory to inhibitory switch in PFC (eCB disruption); ↓upregulation & expression of KCC2 (K+ transporter), Vocalizations ↑in frequency (kHZ). Significance: Delayed development of GABA switch leads to sensorimotor gating deficits, potential autism, epilepsies, schizophrenia-like behavior.	2*	¹¹
Wistar M Adult: 12 days mated to untreated F. Dose: Gavage 2.0 mg/kg/day. Vehicle: EtOH/TritonX100/saline	F1 M/F Pups: PND 28–140, Adolescent, adult	↑Habituation of locomotor activity, Novelty suppressed feeding: ↓latency to begin eating; ↓Visual operant signal. Significance: Impaired operant attention into adulthood	2*	¹¹⁸
SD Juvenile M/F: PND 10–16. Dose: Gavage 10 mg/kg/day. Vehicle: corn oil	F1 M/F Pups: PND 29 & 38, Adolescent	↓Bodyweight gain; High Illumination: ↑entries & time in open arm; Low Illumination: ↑stretch attend posture; ↑head dips; ↓ exploration, ↑frequency of nape attacks; ↑time & frequency play fighting. Significance: Altered social behavior in adolescence.	10*	¹³⁷
C57BL/6J Mice M Pup: PND 5–16 & 5–35. Dose: s.c. 1.0, 5.0 mg/kg/day. Vehicle not stated	F1 M Pup: PND 16 or PND 35 Preweaning, adolescent	Hippocampal cell rearranged CB1R; changes key molecular constituents of mitochondrial respiratory chain; Thinning of pyramidal cell layer; Neurochemical deficits Significance: Developmental deficits from neuronal disorganization, misrouted differentiation & associated pathologies.	1.0	¹¹⁹
Adolescent Treatment
Long-Evans M PND 28 each 3rd day to PND 50. Dose: i.p. 1.5 mg/kg/day. Vehicle: saline/H₂O/Tween80	M: PND 50 or PND 63, Adolescent, adult	Adolescent: Disrupted development of dendritic arbors PFC (pyramidal neurons); Adult: prolonged atrophy in distal apical arbors of PFC neurons; Prematurely pruned dendritic spines attenuated neuroplasticity. Significance: Disrupted PFC neural networks lead to decreased cognitive & emotional dysregulation & affected decision making similar to pathology in human schizophrenia	1.5*	²⁹
Long-Evans F PND 35–75. Dose: i.p. 5.6 mg/kg/day. Vehicle: saline	F: PND 75–160 & 159 to 200 Adult	Adult: ↑CB1R density; Persistent impairment of working memory & task performance. Significance: Long term effects on operant learning	5.6	¹²³
Long-Evans M/F “Puberty Onset” for 14 days. Dose: i.p. 5 mg/kg/day. Vehicle: EtOH/Cremophor/saline	M/F: Day 14 treatment	M/F combined: ↓Total attacks, total pins, percent defense & complete rotation.	5.0 (only dose)	¹³⁸
Wistar M i.p. 1.0 mg/kg/day PND 28–30 →5.0 mg/kg/day alternate days PND 34–52 or PND 60–62 →5.0 mg/kg/day alternate days PND 66-84 or Acute: 5 mg/kg/day: PND 52. Vehicle: Tween80/saline	M: PND 52, 55, 67, 70, 71, 72, 84, 87, 99, 102, 103, 104, Adolescent, adult	Adolescent: ↑Latency to emerge; ↓time in open areas; ↓rearings; ↓novel object preference; ↑memory deficits; alterations in hippocampal structure/function remaining to adulthood. Significance: Hippocampal alterations lead to persistent memory deficits that developed in adolescence	1.0	¹²⁰
SD M/F PND 35–37; 5; 38–41; 10; 42–45. Dose: i.p. 2.5 mg/kg/day, twice/day. Vehicle: EtOH/cremophor/saline	M/F: PND 75: Adult	Adult: ↓ Bodyweight & food intake; ↓CB1R binding & stimulation (NAc, amygdala, VTA, hippocampus); ↓sucrose preference (anhedonia); ↓CREB activation in prefrontal cortex, NAc, hippocampus; ↑ dynorphin (indicates depression). Significance: Disruption of neural circuitry related to emotion and depression during adolescence	5.0	³⁴
SD M PND 35–37; 5; 38–41; 10; 42–45. Dose: i.p. 2.5 mg/kg/day, twice/day. Vehicle: EtOH/cremophor/saline	M: PND 75 Adult	Adult: ↓Radial maze learning; ↓dendritic length in hippocampal dentate gyrus; ↓spine density; ↓NMDA receptors & biomarkers indicating ↓neuroplasticity. Significance: Spatial memory & cognitive deficits	5.0	¹²²
CD1 Mice M PND 28–48; 69–89. Dose: i.p. 3.0 mg/kg/day. Vehicle EtOH/chermophor/saline	PND 49–53 & PND 90–94 Adolescent. PND 90–94 & PND 131–135 Adult	Adolescent: Impaired object recognition/working memory (novel object recognition & discrimination); repetitive/compulsive behaviors (↑percent shredded in nestlet; ↑marble burying); ↓delayed anxiety to move out of the dark; Adult: ↓novel object recognition performance; elevated plus maze ↓ anxiety to venture out. Significance: Behaviors common to those seen in animal schizophrenia models & humans	3.0*	¹²¹
Adult treatment
Long-Evans M. Dose: Acute i.p. 1.0, 1.5, 2.0 mg/kg. Vehicle: detergent/EtOH/saline	∼15 min time increments postdose	↓Attention; ↓hippocampal functional cell types. Significance: Information not likely to be encoded correctly & unlikely to be accurately retrieved or recalled	0.5	¹²⁷
Long-Evans M. Dose: Acute i.p. 0.01, 1.0 mg/kg. Vehicle: Tween80/saline	30 min postdose	↑Trials to achieving reversal task between stimulus & reward; affects c-fos expression associated with negative behavioral effects (orbital limbic & striatal regions in brain). Significance: Effects in orbitofrontal cortex & striatum (potential inelasticity) leading to an inability to perform reversal discriminations	1.0	¹²⁴
Wistar M: 5 days. Dose: i.p. 2.0, 4.0 mg/kg/day. Vehicle: Tween 80/saline	30 min postdose	↓Short-term memory & discrimination affected by eCB increase at the CB1R. Significance: Disrupted CB1Rs is detrimental to memory & cognition	2.0	¹³⁹
Wistar M: i.p. 7 days per dose. Dose: i.p. 1.0, 3.0, 10 mg/kg/day. Vehicle: EtOH/Tween 80/saline	20 min postdose	↓ Body weight; Anxiety measures: ↓time spent in emergence test; ↑ hide time; ↓ open field time; ↓percent open arm time; ↓active time; ↓total social interaction time & distance traveled; Place conditioning: ↓preference for the conditioned side; ↓CB1 R binding in hippocampus, substantia nigra, caudate putamen, cigulate gyrus. Significance: Affected anxiety, learning, memory & social interaction due to disruptions in CB1R binding in critical brain regions	1.0, 3.0, 10	¹²⁹
SD M 2 times/day for 14 days. Dose: i.p. 5.0 mg/kg twice per day. Vehicle: Tween 80/saline	Post terminal dose	↓Performance attention, executive functions, memory, cognition associated with ↓DA in PFC. Significance: Disruption of the cortical dopaminergic pathways lead to cognitive & attention dysfunction	20	¹⁴⁰
SD M: Acute (1 treatment). Dose: i.p. 5.0 mg/kg. Vehicle: OH-β-cyclodextrin/saline	30 min postdose	↑Working memory impairments; ↑DA turnover (DOPAC/DA); ↑NE turnover PFC. Significance: Cognitive impairment	5.0*	¹⁴¹
C57BL/6JArc mice M: 1 or 21 days. Acute & Chronic Doses: i.p. 0.3, 1.0, 3.0, 10 mg/kg. Vehicle: EtOH/Tween 80/saline	Acute & chronic 60 min postdose	Acute & chronic: ↑analgesia & catalepsy; ↓ thermic response & locomotor activity; Anxiety: ↓ distance traveled light/dark; ↓ frequency of entries in elevated + maze; ↓ vertical activity, rearing & head dipping; ↓startle response; ↓passive avoidance/anogenital sniffing, social interaction; ↑latency passive avoidance; ↑prepulse inhibition. Significance: Effect on neurotoxicity (anxiety) occurs after both acute & chronic exposure	1.0	¹²⁶
CD1 mice M. Dose: Acute i.p. 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12, 48 mg/kg/day. Vehicle: EtOH/CremophorEL/saline	30 min postdose	↑Percent time in the open arm in the elevated plus maze; ↓anxiety; ↑percent swim time; ↑closed arm entries. Significance: ↓Anxiety & depression behaviors	0.8	¹²⁵

ACh, acetylcholine; 2-AG, 2-arachidonoylglycerol; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CB1, cannabinoid 1 receptor; CHaT, choline acetyltransferase; CREB, cyclic adenosine monophosphate response element-binding; DA, dopamine; DAGL, diacylglycerol lipase; DOPAC, L-3,4-dihydroxyphenylacetic acid; DRD, dopamine receptor; eCB, endocannabinoid; F, female; GABA, gamma-amino butyric acid; GD, gestation day; GAD67, glutamic acid decarboxylase 67; GLUT, glutamate; H3C, hemicholinium-3; i.p., intraperitoneal; i.v., intravenous; LOEL, lowest-observed-effect level; LTD, long-term depression; M, male; MAGL, monoacylglycerol lipase; NAc, nucleus accumbens; NE, norepinephrine; PENK, preproenkephalin; PFC, prefrontal cortex; PND, postnatal day; PPI, prepulse interval; s.c., subcutaneous; SD, Sprague-Dawley; VTA, ventral tegmental area; ↓, decrease; ↑, increase; →, leads to; *, only one dose was used in the study.

In support of the gestational exposure findings, a meta-analysis was performed on the behavioral effects in animal offspring exposed to ^Δ9THC during gestation and lactation.¹⁴⁸ A compilation and meta-analysis of behavior in offspring from 15 selected studies in Long-Evans, Sprague-Dawley, or Wistar female and/or male rats exposed from mothers exposed via oral, i.v., or s.c. administration indicated significant effects on cognitive, locomotor, and emotional behavior.

Postnatal exposure to ^Δ9THC

Postnatal exposures to young C57BL/6J male mouse pups resulted in behavioral effects from ^Δ9THC treatment at 1.0 mg/kg/day administered s.c.¹¹⁹ This and other studies performed in male and female Wistar rats at 2.0 mg/kg/day (s.c.)¹¹ or 10 mg/kg/day (gavage)¹⁴⁹ included effects on anxiety and neurodevelopmental deficits similar to those seen in autism, epilepsy, and schizophrenia.^11,119,149 Perinatal exposure in children would likely be from nursing, secondhand smoke, or accidental ingestion, causing long-term effects.^37,150 The transfer of cannabis in the milk to nursing babies was shown to affect DA receptors, resulting in hyperactivity, poor coordination, and cognitive function and leading to an increased risk of future drug abuse.^37,150 For example, GABA is primarily excitatory in early development and then it switches to inhibitory postnatally. Disruption of this process in humans may result in neurodevelopmental patterns affecting chronic pain, neuroplasticity, and psychiatric diseases (e.g., autism, epilepsy, and schizophrenia).^11,151 Data indicate that perinatal cannabis exposure increases the risk of future drug use.¹⁵²

Adolescent exposure to ^Δ9THC

Exposure to ^Δ9THC i.p. in Long-Evans and Wistar male rats throughout adolescence at low doses (1 or 1.5 mg/kg/day) showed disrupted neural development in the PFC and hippocampus resulting in effects on neuroplasticity, cognition, social interactions, memory and others.^29,120 Similar effects (i.e., increased: CB1R density, anxiety, learning deficits, anhedonia) were observed at higher doses (2.5–10 mg/kg/day) in Long-Evans females, male and female Sprague-Dawley rats and male CD1 mice receiving various dosing regimens (Table 2).^{120–123,153Δ9}THC treatment in adolescents disrupted development of brain areas (e.g., PFC) associated with adverse behaviors like schizophrenia in humans, which often occurs in adolescence.¹⁵⁵ Adolescence is a stage of peak eCB (2AG and AEA) and CB1R expression.¹⁵⁶ The brain is still developing and is at heightened risk for disruption of normal neurodevelopmental processes.^157,158 Where pre- or postnatal exposures may be involuntary in developing young, adolescence is where preteens and teens may begin to experiment with cannabis on their own.¹⁵⁹ Vaping cannabis has become one of the most preferred methods of consumption, that will not only increase the concentration of ^Δ9THC but also potentially increase exposure to residues of pesticides used on cannabis crops.^99,160–162 Cannabis use in adolescents greatly increases the risk of psychosis by 3–4-fold and has been shown to lower the age of schizophrenia onset.^163,164 Further, adolescent cannabis use will increase the probability of future drug use,¹⁶⁵ as shown by evidence from animal and epidemiological studies.^152,166,167

Adult exposure to ^Δ9THC

Acute adult effects in Long-Evans male rats as well as C57BL/6Arc and CD1 male mice showed behavioral effects (attention and learning, decreased anxiety and locomotor activity) at low ^Δ9THC doses (i.p.: 0.25, 0.8, or 1.0 mg/kg/day; Table 2).^124–127 Notably, these studies used 2–8 treatment levels and could therefore establish a dose–response relationship. C57BL/6J male mice treated at 10 mg/kg/day also experienced a decreased thermic response and increased catalepsy and analgesia.¹²⁶ This study demonstrated the “cannabinoid tetrad”: increased catalepsy, hypomobility, hypothermia, and antinociception.¹²⁸ At the low acute doses, the animals showed decreased anxiety; but at higher doses graduating from 1 to 3 to 10 mg/kg/day at 7-day intervals, the animals had increased anxiety measures with both acute and chronic exposures in male Wistar rats.¹²⁹ Human studies also showed that cannabis use versus nonuse was associated with an earlier onset of psychoses, death by suicide, depression, mania, anhedonia, cognitive deficits, and anxiety/paranoia as well as brain effects (decreases in glutamine, affected DA, and decreased hippocampal volume (systematic review).¹⁶⁸ This review also reported associated harmful effects of exposure on driving, stroke, pulmonary function, vision, and negative drug-drug interactions. With cannabis legalization, it is likely that there will be more health-related deficits and an increased need for public and clinical policy changes. Table 2 lists the lowest-observed-effect levels (LOELs) reported from each in-vivo study (mg/kg/day).

Nonhuman primate exposure to ^Δ9THC

Studies in nonhuman primates have been performed in pregnant animals. Rhesus macaques were fed ^Δ9THC in a cookie at 2.5 g/7 kg at gestation day (GD) 0–155.¹⁶⁹ There were decreases in the amniotic fluid volume throughout pregnancy and decreased placental perfusion (oxygen availability decreases) accompanied by increased placental microinfarctions. In addition, there were significant changes in the RNA signature sequences in the placental transcriptome. These data indicate that disruptions in vascular development and angiogenesis affect the offspring through decreased testes weights and relative heart weights. Adult male rhesus macaques were treated with ^Δ9THC in a cookie at 0.5 mg/7 kg/day (1–70 days), 1.0 mg/7 kg/day (71–140 days), and 2.5 mg/7 kg/day (141–210 days). At 210 days, there were dose-related decreases in testicular and epididymal weights.¹⁷⁰ Follicle-stimulating hormone, luteinizing hormone, and prolactin were increased, and total testosterone and estradiol were decreased. These effects indicate potential disruption of the hypothalamus–pituitary–gonadotropin axis, impacting testicular function.¹⁷¹ In another study, adult female rhesus macaques were treated with ^Δ9THC in a cookie at 0.5 mg/7 kg/day (1–3 weeks), 1.0 mg/7 kg/day (4–6 weeks), 2.0 mg/7 kg/day (7–9 weeks), and 2.5 mg/7 kg/day (10–12 weeks). At 12 weeks, the animals showed increases in menstrual cycle length and increased follicle-stimulating hormone concentrations, another indication of hypothalamus–pituitary–gonadotropin axis disruption.¹⁷¹ The disruptions in hormonal balance, menstrual cycle, and ovulatory function would likely affect fecundity.¹⁷²

^Δ9THC-associated effects in humans

A review by Frau and Melis¹⁷³ provides evidence showing that in utero, transplacental ^Δ9THC exposure deregulates the mesolimbic dopaminergic system in males, potentially predisposing them to schizophrenia. Prenatal exposure in humans can act to prime the sensorimotor gating development in the brain, primarily in the VTA region associated with the dopaminergic system. Subsequent environmental exposures such as ^Δ9THC or other stressors can lower the threshold to initiation of psychotic-like effects.¹³⁴ In addition, ^Δ9THC exposure to infants during breastfeeding can continue more than 6 weeks after the last maternal consumption, potentially affecting brain development.^9,38,142,174 Monfort, Ferreira, Leclair, and Lodygensky²² have described the pharmacokinetics of cannabinoid exposures during pregnancy, in infants, and during breastfeeding. While consumption may be due to depression, anxiety, nausea, or pain, data indicate that there are significant irreversible risks to neuronal development in fetuses, neonates, and the developing young.²² Data also support the increased risks of dysregulated glucose-insulin measurements as well as obesity in children after maternal use of cannabis during pregnancy.¹⁷⁵

Although ^Δ9THC (cannabis) is not federally legal in the United States, acute and repeated human exposure to ^Δ9THC is regulated by the European Food Safety Authority.¹⁷⁶ Human data were used by this agency to establish a lowest-observed-adverse-effect level (LOAEL) for an administered ^Δ9THC exposure of 2.5 mg/kg/day (corresponding to an internal dose of 0.036 mg/kg/day). Applying an uncertainty factor of 3 to extrapolate from a LOAEL to a no-observed-adverse-effect level (NOAEL) and 10 for intraspecies differences produced 1 µg/kg/day (acute reference dose: ARfD = [0.036 mg/kg/day ÷ 30] = 1 µg/kg/day). However, it is evident from gestational treatment in Table 2 that offspring experienced neurodevelopmental effects related to motivation/reward, stress response, and increased sensitivity to opiate reward in adulthood at 0.15 mg/kg/day.^112,114 Establishing an ARfD would require the same uncertainty factors in addition to an interspecies default of 10 ([LOAEL 0.15 mg/kg/day ÷ 3 = NOAEL 0.05 mg/kg/day] ÷ [10 interspecies × 10 intraspecies]) = 0.5 µg/kg/day.^177–179 Gestational exposure to ^Δ9THC may need a different ARfD than that of adults, since effects occur at very low doses. This is especially critical to re-evaluate because the low-dose animal studies used only one dose, and there were no doses below 0.15 mg/kg/day in which effects might also be seen in developing fetuses.

CBD-associated mechanisms

While it can make up as much as 40% of cannabis extract,¹⁸⁰ CBD has been purified in products for use by people and even their pets. CBD is one of the most actively studied therapies for a broad spectrum of neurological, inflammatory, and mental diseases because of its efficacy, low toxicity, and availability (e.g., over the counter and online order). The exact mechanism for the therapeutic effects is still under investigation,^42,181 but the proposed MOA for CBD indicates several targets associated with neuroprotection (Fig. 2¹⁸² and Table 3).^86,183,184 Like ^Δ9THC, CBD has effects on many interacting targets, and there is evidence for direct and indirect CBD actions on inflammatory and neurological parameters.¹⁸⁰

The cannabidiol (CBD) mechanism of action includes: (1) agonistic activity toward the transient receptor potential vanilloid type 1 (TRPV1), the peroxisome proliferator activated receptor ɣ (PPARɣ), and the serotonin<sub>1A</sub> (5-HT<sub>1A</sub>) receptor; (2) antagonist activity at the G-protein coupled receptor GPR55; (3) antagonist to CB1 and CB2Rs in addition to acting as a reverse agonist and negative allosteric modulator; (4) antagonist of FAAH leading to increased anandamide (AEA), which goes on to activate the CB1, CB2, and TRPV1 receptors.; (5) direct action on the GABA<sub>A</sub> receptor (also influenced by AEA), leading to neuroprotection; (6) increased mitochondrial activity leading to antioxidant and anti-inflammatory action.

Fig. 2 The cannabidiol (CBD) mechanism of action includes: (1) agonistic activity toward the transient receptor potential vanilloid type 1 (TRPV1), the peroxisome proliferator activated receptor ɣ (PPARɣ), and the serotonin_1A (5-HT_1A) receptor; (2) antagonist activity at the G-protein coupled receptor GPR55; (3) antagonist to CB1 and CB2Rs in addition to acting as a reverse agonist and negative allosteric modulator; (4) antagonist of FAAH leading to increased anandamide (AEA), which goes on to activate the CB1, CB2, and TRPV1 receptors.; (5) direct action on the GABA_A receptor (also influenced by AEA), leading to neuroprotection; (6) increased mitochondrial activity leading to antioxidant and anti-inflammatory action.

Overall CBD has anti-depressant, anti-anxiety, and anti-inflammatory effects. Figure adapted with permission: Copyright © 2018.¹⁸² FAAH, fatty acid amide hydrolase; GABA, gamma-aminobutyric acid; GPR55, G protein-coupled receptor 55; ROS, reactive oxygen species.

Table 3

In-vivo and in-vitro examples of neuroprotective effects of CBD in different neurological diseases¹⁸³

Model	CBD dose	Treatment	Biological/pharmacological effect	Neurological disease
Neuroprotection through activation of A_2ARs
SJL/J mice: F	5.0 mg/kg, i.p.	Days 1–7 post infection	Microglia activation attenuated, downregulating the expression of VCAM1, CCL2 and CCL5 & proinflammatory cytokine IL1β. CBD improved motor deficits in the chronic phase of the disease	Multiple sclerosis
Newborn C57BL6 mice: M/F	0.1–1,000 µM	15 min pre-incubation	↓Acute brain damage & apoptosis; ↓glutamate concentration, IL6 & expression of TNFα, COX2, and iNOS	Hypoxic-ischemic brain damage
Primary rat microglial & N13 microglial cells & C57Bl/6 mice: M/F	20 mg/kg, i.v.	1/day for 7 days; 3 days/week for 2 weeks	Inhibited ATP-induced intracellular Ca²⁺ increase in cultured N13 & primary microglial cells and A_2A receptors may be involved in this mechanism. In vivo: ↓gene expression of proinflammatory cytokine IL6 & prevented cognitive impairment induced by βA	Alzheimer’s disease
Sabra mice: F	5.0 mg/kg, i.p.	28 days	↓Hippocampal TNFα-R 1 gene expression but ↑expression of the BDNF gene. Indirect activation of A_2AR, ↑cognitive & motor function in rats with hepatic encephalopathy.	Hepatic encephalopathy
Neuroprotection through the activation of the 5-HT_1A
MCA occlusion mice: M	3.0 or 10 mg/kg, i.p.	Before & 3 h after damage	CBD significantly ↓infarct volume induced by MCA occlusion through 5-HT_1A receptor	Cerebral ischemia
Swiss mice: M	5.0, 15, 30, or 60 mg/kg, i.p.	30 min before receiving drugs to induce catalepsy	CBD pretreatment ↓catalepsy in a dose-dependent manner, through the 5-HT_1AR	Striatal disorders
Swiss mice: M	15–60 mg/kg or 60 nmol, i.p.	30 min before or 2.5 h after receiving the drugs to induce catalepsy	CBD pretreatment ↓catalepsy in a dose-dependent manner, through the 5-HT_1AR	Striatal disorders
Wistar Kyoto rats: M	100 mg/kg	60 min before seizure induction	CBD significantly mitigated PTZ-induced seizure	Seizure disorders
Adult Wistar rats: M	0.1–1.0 mg/kg & 5.0 mg/kg, i.p.	Acute treatment + cumulative injections every 5 min & repeated at 5 mg/kg/day for 7 days	CBD protected nerve injury-induced deficits in dorsal raphe nucleus 5-HT neuronal activity & exerted antiallodynic effects by TRPV1 activation & anxiolytic properties through 5-HT_1A receptor activation	Allodynia & anxiety
Sabra mice: F	5.0 mg/kg, i.p.	28 days	CBD, by 5-HT_1AR activation, ↑cognition & motor function, impaired by bile-duct ligation. CBD ↓neuroinflammation, ↑BDNF gene expression & ↓TNFαR 1 gene expression in hepatic encephalopathy model	Hepatic Encephalopathy
Sabra mice: F	5.0 mg/kg, i.p.	Single acute dose	CBD ameliorated cognitive deficits & locomotor activity; restored brain 5-HT levels & improved liver function	Hepatic Encephalopathy
C57BL/6J mice: M	30 mg/kg/day, i.p.	7 days	↑Time spent interacting; ↓psychotic-like behaviors acting through 5-HT 1A receptors	Schizophrenia
Neuroprotection by antagonistic activation of GPR55
Scn1a mutant mice (DS model): M/F	10, 20, 100, or 200 mg/kg/day	Twice/day for 7 days	Acute CBD ↓thermally induced seizures & ↓spontaneous seizure rate. Low doses ameliorated autism-type social interaction deficits in genetically induced DS model, ↑GABA inhibitory transmission impaired in DS mediated by GPR55	DS
Adult C57BL/6 mice: M	5.0 mg/kg	5 days/week, 5 weeks	↓Density of microglial cells in the cell body. In the haloperidol-induced catalepsy model, through GPR55-activation.	Parkinson’s disease
C67BL/6 mice M/F	5.0–10 & 50 mg/kg	Increasing doses from 5.0 to 10 mg/kg 3 times/week, or daily, at 50 mg/kg, for 23 days	EAE disease ameliorated (all doses), ↓encephalitogenic cell vitality, ↓levels of IL6, production of ROS, ↓apoptosis & GPR55R in CNS	EAE disease
Neuroprotection through activation of the TRPV receptors
Wistar rat: M	10 mg/kg, i.p.	2 h after the induction of model	CBD inhibited carrageenan-induced hyperalgesia by desensitization of TRPV1R	Hyperalgesia
hPBMECs & hCMEC/D3 Cells^a	0.1, 0.3, 1.0, 3.0, 10, 15 µM	7 or 24 h of incubation	Dose-related ↑in intracellular Ca²⁺ through activation of TRPV2 enhanced cell proliferation, cell migration & tubulogenesis in human brain endothelial cells.	—
U87MG Human glioblastoma cell line	10 µM	Cells treated with different CBD doses 1 day or co-treated with CBD 10 µM & chemo drugs 6 h	TRPV2 activation & ↑Ca²⁺ improved chemotherapy drug action by enhancing absorption & ameliorating cytotoxic activity in human glioma cells	—
Human gingival mesenchymal stem cells	5 µM	24-h incubation	TRPV1 desensitization promoted the PI3K/Akt pathway^b signaling, which can reduce Alzheimer hallmarks	Alzheimer’s disease
Neuroprotection through the activation of the PPARɣ
SH-SY5Y^APP+ cells	10⁻⁹–10⁻⁶ M	24 h	↓Expression of amyloid precursor protein & its ubiquitination, leading to ↓Aβ & neuronal apoptosis. Effects mediated by PPARɣ activation	Alzheimer’s disease
Primary rat astrocytes & SD rat: M	10⁻⁹–10⁻⁷ M: in vitro; 10 mg/kg: in vivo, i.p.	15 days	In vitro: Dose response ↓ in Aβ mediated through inhibition of NF-кB; Aβ-induced neuronal damage led to ↓gliosis & glial fibrillary acidic protein. Effects exerted through PPARɣ activation	Alzheimer’s disease
Hippocampal slices from C57Bl/6 mice	10 µM	30 min before addition of Aβ	Improved synaptic transmission & long-term potentiation in the hippocampus slice of C57BL/6 mice, protecting it from cognitive deficits induced by Aβ 1–42. CBD effects exerted through interaction with PPARɣ	Alzheimer’s disease
Newborn C57/BL6 & Swiss mice primary microglial cultures: M/F	60 mg/kg: in vivo, i.p.; 10 µM: in vitro	2 injections/day 30 min prior to haloperidol: 21 days	Dyskinesia prevented after induction haloperidol. ↓Oxidative stress in corpus striatum, ↓activation of microglial, inflammatory cytokine (e.g., IL1β and TNFα), ↑anti-inflammatory cytokine IL10. CBD affects PPARɣ actions on lipopolysaccharide-stimulated microglial cells	Tardive dyskinesia
Adult C57/BL6 mice: M	15, 30, or 60 mg/kg, i.p.	15 min before L-DOPA administration for 3 days	CBD did not prevent L-DOPA-induced dyskinesia. Cotreatment of CBD + capsazepine, acting through CB1R & PPARɣ, ameliorated dyskinesia.	Parkinson’s disease
Human brain microvascular endothelial cell/human astrocyte co-cultures	100 nM, 1.0 & 10 µM	Before or directly after induction of ischemic damage	10 µM prevented enhanced BBB permeability after ischemic damage induced by oxygen-glucose deprivation, through by activating PPARɣ & 5HT_1AR.	Ischemic stroke
Neuroprotection through positive allosteric modulation of GABA_A receptors
Surgical human DS & TSC cortical tissue in Xenopus oocytes	5.0 µM	Pre-incubation of cells 10 s before co-application of GABA & CBD	Positive modulation of GABA_AR, ↑amplitude of GABA-evoked current in brain tissues of patients with DS &TSC	DS & TSC
Scn1a+/- mice (M/F) & Xenopus oocytes expressing GABA_A receptors	in vivo 12 or 100 mg/kg, i.p.; in vitro 10 µM	In vivo: CBD administered i.p. 45 min before CLB. In vitro: CBD (10 µM) co-applied with GABA, for 60 s	↑CLB concentration & active metabolite N-CLB in plasma & brain. Co-administration ↑anticonvulsant effect by enhancing the activity of the GABA_A receptor	DS

^ahCMEC/D3 cells: Human hematopoietic stem-cell-derived cells (HBLECs) and human primary brain microvascular endothelial cells (hPBMECs) used in BBB models. ^bPI3K/Akt pathway: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt kinase phosphorylation involved in the cell cycle is decreased in the Alzheimer’s brain (associated with amyloid-β and tau pathologies).¹⁸⁴ βA, β-amyloid; A2AR, adenosine 2A receptors; ATP, adenosine triphosphate; BBB, blood–brain barrier; BDNF, brain-derived neurotrophic factor; CBD, cannabidiol; CB2R, cannabinoid receptors type 2; CCL-2, chemokine ligand 2; CCL-5, chemokine ligand 5; CLB, clobazam; N-CLB, N-desmethylclobazam; CNS, central nervous system; COX2, cyclooxygenase-2; DS, Dravet syndrome; F, Female; GABA, ɣ-aminobutyric acid; GPR55, G-coupled protein receptor 55; hPBMECs, human primary brain microvascular endothelial cells; IL1β, interleukin 1β; IL6, interleukin-6; IL17, interleukin-17; iNOS, inducible nitric oxide synthase; L-DOPA, l-3,4-dihydroxyphenylalanine; M, male; MCA, middle cerebral artery; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PPARɣ, peroxisome proliferator activated receptor-ɣ; PTZ, pentylenetetrazole; ROS, reactive oxygen species; Scn1a+/-, heterozygous loss of function SCN1A; SD, Sprague-Dawley rat; SH-SY5Y^APP+, SH-SY5Y cells transfected with the amyloid precursor protein; TNFα, tumor necrosis factor α; TSC, tuberous sclerosis complex; TRV1 or 2, transient receptor potential vanilloid type 1 or 2; VCAM1, vascular cell adhesion molecule-1. Table adapted with permissions: Creative Commons — Attribution 4.0 International — CC BY 4.0.^86,183

Glial cells

CNS connective tissue (e.g., macroglia: astrocytes and microglia) consists of nonneuronal cells that link neuronal cells to the blood supply (blood–brain barrier), regulate blood flow to the brain, and regulate neurotransmission (macroglia) or serve as macrophages to mount immune responses in the brain (microglia).¹⁸⁵ When neuronal injury occurs, astrocytes can signal microglia to initiate an immune response; however, when the immune response becomes unbalanced, neuronal injury will occur.¹⁸¹ CBD can decrease the microglial immune response to injured dopaminergic neurons in diseases like Parkinson’s disease, and it increases the recruitment of astrocytes to promote neuronal regeneration through brain-derived neurotrophic factor (BDNF) (Table 3).

Adenosine receptor 2A (A_2AR)

Adenosine acts at a G protein-coupled receptor (A_2AR) on neuronal membranes to suppress immune responses due to inflammation or cell stress. CBD serves as an agonist to decrease adenosine reuptake, thereby increasing adenosine signaling and decreasing neuroinflammation.^186,187 CBD exposure decreases proinflammatory cytokine interleukin (IL)1β, microglial activity, tumor necrosis factor-alpha (TNFα), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) activity in the brain (Table 3). These pathways have been shown to improve the effects of multiple sclerosis, hypoxic-ischemic brain damage, Alzheimer’s disease, and hepatic encephalopathy.¹⁸³

5-HT receptors

The dorsal raphe nucleus (DRN) is the primary serotonergic center (5-HT) in the brain where GCPR 5-HT_1A receptors are expressed. Receptor stimulation inhibits voltage-gated Ca²⁺ channels, activates K⁺ channels, and inhibits neurotransmission in the DRN.^44,188 CBD has an anxiolytic effect by acting through 5-HT_1A receptor in male Wistar rats, previously stressed by foot shocks or restraint, but it can also induce anxiogenic behaviors in rats experiencing contextual fear conditioning,^182–190 perhaps by serving as an agonist at the 5-HT_1A receptor.¹⁸⁸ Acting on the serotonergic system, CBD is associated with improved locomotor activity (after striatal damage), cognition, cerebral ischemia, seizure disorders, and hepatic encephalopathy (Table 3).¹⁸³ Through the 5-HT1A receptor, CBD is associated with antiepileptic, anticataleptic, neuroprotective, antiemetic, anxiolytic, antidepressant, antipsychotic, and analgesic effects.^86,191–195 Others have also indicated that CBD acts via a negative allosteric mechanism in DRN somatodendritic 5-HT_1A receptors that does not require CB1, 5-HT_2A, or GABA_A receptors.^86,186

CB1Rs and CB2Rs

CBD at the CB1Rs regulate excitotoxicity by inhibiting glutamate release to the NMDA receptors and normalizing glutamatergic activity. CBD acts to increase the blood supply to areas after ischemic incidents by decreasing endothelial-derived endothelin-1 or nitric oxide to increase vasoconstriction.¹⁹⁷ Neurodegeneration occurs with activation of microglial cells (immune cells in the brain); however, CB1R activation by CBD leads to a decrease of TNFα and IL12 and an increase of IL10. Activation of CB2 then decreases the proliferation and migration of microglial cells while decreasing TNFα by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NFкB; Table 3).^198,199 The anti-inflammatory action of CBD has been shown to improve neuronal damage from ischemic stroke, Tardive dyskinesia, and Parkinson’s disease.

FAAH

CBD can act indirectly at the CB1R through inhibition of FAAH and the AEA transporter, leading to increased AEA and activation of CB1R.^200,201 Increased CB1R agonism leads to decreased eCB degradation and transport (Table 3).

TRPV1

TRPV consists of a vanilloid channel on the plasma membrane, considered by some to be a CB3R,⁵¹ that induces neuropeptide release associated with pain perception, neuroinflammation, and body temperature regulation.²⁰⁰ CBD at TRPV-1 channels leads to increased Ca²⁺ levels, resulting in desensitization and subsequent decreased pain. TRPV1 binding decreases microglial activation and migration as well as oxidative stress (Table 3). In addition, CBD can increase AEA levels by inhibition of FAAH.²⁰² However, AEA and CBD are both TRPV1 channel agonists. TRPV1 channel activation by CBD presynaptically increases glutamate release in the brain, which may serve to counteract/antagonize the inhibitory action of CB1R binding by CBD on colocalized glutamatergic neurons. TRPV1 activation by CBD agonism can increase the PI3K/Akt pathway signaling to decrease the incidence of hallmarks of Alzheimer’s disease.

G-coupled protein receptor 55 (GPR55)

GPR55 binding protects against excitotoxicity potentially through GABA_A receptor. CBD, as an antagonist, decreases GPR55 activation in the CNS to regulate such processes as neuropathic pain and antiepileptic activity.²⁰³ CBD has a high affinity for GPR55, resulting in a decreased glutamate release in the hippocampus, thus causing anti-convulsive effects, also seen in human subjects.¹⁸⁰ Moreover, the use of CBD has been shown to result in improved Parkinson’s disease and Dravet syndrome (DS) symptoms (Table 3).^183,204

Peroxisome proliferator-activated receptor gamma (PPARɣ) receptors

CBD is an agonist of PPARɣ, a nuclear receptor and ligand-inducible transcription factor that produces anti-inflammatory and antioxidative effects.¹⁹⁹ PPARɣ modulates inflammation by inducing ubiquitin-proteasomal degradation of p65, resulting in inhibition of proinflammatory gene expression of cyclooxygenase (COX2) and proinflammatory mediators (e.g., TNFα, IL1β, and IL6) in addition to inhibition of NFкB-mediated inflammatory signaling. CBD agonist activity with PPARɣ also contributes to the inhibition of TNFα, IL1β, and IL6 transcription to prevent NFкB signaling, and it also produces antioxidant properties.^198,199 It increases eCBs by antagonist activity at CB2Rs, and the eCBs then act as PPARɣ agonists to promote anti-inflammatory and antioxidant actions. Furthermore, Alzheimer’s disease has been demonstrated to be improved via the PPARɣ-mediated protective effects of CBD (Table 3).

GABA_A receptors

As the main inhibitory neurotransmitter in the CNS, GABA disruption is associated with neurological diseases, including cognitive deficits, drug addiction, chronic stress and anxiety, epileptic disorders, and Huntington’s disease.^180,205 CBD stimulates GABAergic neurotransmission, meaning that the inhibitory neurotransmission and frequency are increased.²⁰⁶ Seizure frequency, duration, and severity were reduced in addition to increased social behaviors in a mouse model of DS and other diseases after CBD treatment. In addition, overexcitation in the dentate gyrus of the hippocampus was decreased through CBD effects on GABA_A receptors.²⁰⁶ Therefore, with CBD bound to the GABA_A receptor, anticonvulsant and anxiolytic actions are seen in the CNS. Moreover, since CBD does not bind competitively with the benzodiazepine receptor, it is potentially useful in patients resistant to benzodiazepines, which is the standard antiseizure treatment (Table 3).²⁰⁷

CBD-associated neuroprotection in animal studies

CBD has shown neuroprotective effects in animal models with several neural-associated disease states (Table 3).^{86,181,183,208} The areas studied have focused mainly on neuroprotection and treatment of brain-related diseases (e.g., multiple sclerosis, Alzheimer’s disease, and schizophrenia), rather than effects on other areas of the body (e.g., local pain). CBD at doses from 5.0 mg/kg/day in rodents has many beneficial effects (Table 3). Note that doses administered in vivo were by i.p.; therefore, CBD is more slowly absorbed and subject to local metabolic processes prior to entering the blood stream, as would occur with oral exposure.^107,111Table 3 indicates pathways specifically shown to be associated with CBD exposure.

CBD neuroprotection in human studies

The neuroprotective effects of CBD observed in animal studies are supported by observations in human subjects. CBD is well tolerated in children and adults and has a broad spectrum of therapeutic benefits to help with significant neurological disease states,²⁰⁹ including neurological damage and disorders, brain tumors, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, neuropathic pain, and childhood seizures (e.g., Lennox-Gastaut syndrome and DS).^180,210 Additionally, synthetic forms of CBD have been used to treat drug-resistant epilepsies in children (age ≥2 and older) (Lennox-Gastaut syndrome or DS).²¹⁰ Epidiolex/Epidyolex (>99% CBD) is approved by the United States Food and Drug Administration and the European Medicines Agency to treat these diseases.²¹¹ The benefits of CBD also have been shown in human subjects to treat anxiety, depression, post-traumatic stress disorder, and obsessive-compulsive disorders;^212,213 furthermore, it has demonstrated antipsychotic properties in those with schizophrenia.²¹⁴ A few examples of CBD affecting neurological diseases are listed in Table 4 (review).⁴²

Table 4

Neuroprotection for Parkinson’s disease initiated with cannabidiol treatment

CBD target	Biological effect
CBD neuroprotection in Parkinson’s disease (review)⁴²
CB1 activation	↓Microglial activation and microglial NADPH oxidase expression; ↓Production of proinflammatory agents (IL1β, TNFα, iNOs, COX2); ↓Dopaminergic neuronal damage; ↓Excitotoxicity (↓glutamate release); ↓ROS and lipid peroxidation
CB1 antagonism	↑Astrocyte activation in substantia nigra pars compacta
CB2 activation	↓Microglia number and production of proinflammatory agents (IL1β, TNFα, iNOs, nitric oxide); ↓Dopamine depletion; ↓Myeloperoxidase-positive astrocytes; ↑Antioxidant enzyme activity and antioxidant agents
MAGL inhibition	↓Microglia and astrocyte number; ↑CB2 activation; ↑GDNF
FAAH inhibition	↑Motor activity; prevents excitotoxicity by inhibiting glutamate release due to neuroinflammation; ↓Protein carbonylation; ↓ROS and lipid peroxidation
PPARɣ activation	↓ROS
CBD neuroprotection in Huntington’s disease (review)⁴²
CB1 activation	↓Excitotoxicity (↓glutamate release)
CB2 activation	↓Reactive microglial cell number; ↓Production of proinflammatory agents (TNFα); ↓ROS and nitric oxide; ↑Production of neurotrophins & anti-inflammatory mediators (IL10, IL1 antagonist)
Phytocannabinoid structure	↓ROS (phenolic structure acts as an ROS scavenger)
PPARɣ activation	Interference with the NFκB signaling pathway; Induction of antioxidant enzymes
CBD neuroprotection in Alzheimer’s disease (review)⁴²
PPARɣ activation	↓Apoptosis during neurodegeneration; ↓Astrocyte activation; ↓Expression of proinflammatory cytokine IL1β and iNOS (↓neuroinflammation); ↓Amyloid plaque and inflammation
CB1 activation	↓Amyloid β-induced memory impairment
CB2 activation	↓Proinflammatory mediators from microglial cells and astrocytes; ↓Neuroinflammation

CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; COX2, cyclooxygenase 2; FAAH, fatty acid amide hydrolase; GDNF, glial cell-derived neurotrophic factor; IL, interleukin; iNOS, inducible nitric oxide synthase; MAGL, monoacylglycerol lipase; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; TGF, transforming growth factor; TNF, tumor necrosis factor.

Parkinson’s disease

The hallmark of Parkinson’s disease is the accumulation of α-synuclein and the degeneration of dopaminergic neurons in the SNa in addition to motor alterations (bradykinesia, resting tremors, rigidity, and postural instability), depression, and dementia (review).⁴² Improvement in the disease by CBD occurs via numerous pathways acting through the eCBS (e.g., CB1Rs, CB2Rs, FAAH, and MAGL) to modulate excitotoxicity, dopaminergic neuronal degeneration through inflammation, and microglial inhibition (Table 4).^{43,202,215–217} Importantly, CBD has been used to improve the effects of Parkinson’s disease in human subjects (review).²¹⁸

Huntington’s disease

Huntington’s disease is an autosomal-dominant neurodegenerative disease that is progressive, leading to degeneration of striatal GABA and dopaminergic neuronal destruction in the globus pallidus.⁴³ CB1R activation by CBD in the striatum can inhibit glutamatergic transmission to protect damaged neurons and serve as an antioxidant (Table 4).^{43,217,219,220}

Alzheimer’s disease

CBD has been shown to decrease or block hyperphosphorylation of tau protein, acetylcholinesterase activity, oxidative stress, apoptosis, neuroinflammation, gliosis, and deposition and expression of beta-amyloid (βA).²¹⁰ The mechanism is associated with selective activation of PPARɣ, resulting in increased clearance of βA peptides through autophagy in the hippocampus, ubiquitination of amyloid precursor proteins, and decreased βA deposition (Table 4).^43,210

CBD-associated toxicity

Since it is not considered to be intoxicating, compared to ^Δ9THC, CBD has been widely used for medicinal purposes and is of great interest to medical communities.¹⁷ While CBD use has increased in humans for a plethora of conditions, little is known about the potential for risks from consumption during pregnancy or in children using CBD to treat epilepsy.^17,221 The effects of CBD on brain development in utero are not well understood; however, C57BL6/J dams treated with 3.0 mg/kg s.c. GD 5-18 had pups with sex-specific behavioral effects (Table 5).^{15–17,23,24,183,222,223,228}, The male pups showed higher body weights, and there were effects on ultrasonic vocalizations (both sexes), homing behavior, and decreased motor and discriminatory abilities (females). These findings indicate that CBD has effects on psychopathology after in-utero exposure at 3.0 mg/kg/day and may not be as safe as previously considered when consumed during pregnancy.

Table 5

Neurotoxic, behavioral, and reproductive effects from CBD treatment during development in animal studies

Animal strain sex/duration/dose/vehicle	Day tested	Effects	LOEL (mg/kg/day)	Reference
Gestational treatment
In-vitro C57Bl/6J mouse whole embryos. 6 somite embryos for 24–30 h of culture. Dose: 0, 15, 30 µM CBD. Vehicle: EtOH	24–30 h	No effects on embryo growth. ↓cranial neural tube closure 15, 30 µM. Significance: Adverse effects on brain development in vitro	15 µM	²³
C57Bl/6J mouse M/F: GD 5–18 S.C. Dose: 0, 3 mg/kg/day. Vehicle: Cremophor EL, EtOH, saline	PND 10 and 13	10d: Mean USV duration ↓ (M) and frequency ↑ (F); PND 10, 13, 16, 19, 22: ↓body weight (M); Syllabic repertoire of sound communication sex specific; ↓Homing behavior: Distance moved, velocity, movement distance moved from nest (F). Significance: Adverse neuronal development in vivo	3 mg/kg/day (only dose tested)	²⁴
Postnatal treatment
In Vitro Wistar primary neonatal (PND 2) rat cerebral cortices (astrocytes + neurons) 1–24 h. Dose: 0, 0.5, 1, 5 µM CBD. Vehicle: EtOH	24 h	Neuron: All doses tested: Viability ↓ LDH ↑ at ≥0.1 µM; Only 0.1 µM tested: Change in mitochondrial membrane potential, ↑ATP depletion & caspase 4/7 activation, ↑apoptosis & chromatin condensation; ↓dendrite length; Astrocytes: All doses tested: Viability ↓ LDH ↑ at ≥0.5 µM; Only 0.5 µM tested: dysregulated mitochondrial membrane potential, ↑ATP depletion & caspase 8, 9, 4/7 activation, ↑apoptosis & necrosis. Significance: Cytotoxic to neurons and astrocytes in vitro	Neurons: 0.1 µM; Astrocytes: 0.5 µM	¹⁷
In vitro 18-week-old human M: Sertoli cells mouse sertoli cell line. Dose: Human: 7, 8, 9, 10 µM. Mouse: 10, 12.5, 15, 17.5, 20 µM. Vehicle: DMSO	24 h	Human & Mouse: ↑Cytotoxicity & cell senescence; ↓DNA replication & DNA repair; disruptions in cell-cycle related genes; ↓Cell viability; inhibition of G1/S phase cell cycle transition; ↓mRNA for Wilms’ tumor 1 biomarker. Significance: Adverse effects on human Sertoli cells in vitro	Human: 7.0 µM; Mouse: 10 µM	²²⁸
Adolescent treatment
Swiss mice M: PND 21–55 (4 spermatogenic cycles), gavage. Dose: 0, 15 & 30 mg/kg/day. Vehicle: Sunflower oil	PND 90	↓Testosterone (30 mg/kg/day); ↓spermatogenesis (≥15 mg/kg/day); ↑sperm with head abnormalities & cytoplasmic droplets (≥15 mg/kg/day); affected seminiferous tubule morphology (≥15 mg/kg/day). Significance: Disrupted sperm development likely affected fertility	15 mg/kg/day	²²³
Swiss Mice M: PND 21–55 (4 spermatogenic cycles), gavage. Dose: 0, 15 & 30 mg/kg/day. Vehicle: Sunflower oil	PND 90	Germinal epithelium stages disrupted & seminiferous tubule dysmorphology during spermatogenesis (≥15 mg/kg/day); ↑malonaldehyde & ↓sperm motility, super oxide dismutase & catalase at 30 mg/kg/day; ↑abnormal acrosome reaction & sperm velocity (≥15 mg/kg/day). Significance: Potentially affected fertility & ↑oxidative stress	15 mg/kg/day	¹⁵
Adult CBD treatment
Wistar rat: 1 treatment (M/F) or 4 days (F). Dose: 0, 0.3, 3, 30 mg/kg. Vehicle: Not stated	F: pro- and late diestrus 1 h/4 d; M: 1 h	Acute: Late diestrus ↑entries into & time spent in open arms EPM (0.3 mg/kg/day F; 3.0 mg/kg/day M); 4-day F: Late diestrus ↑entries & time spent into open arms EPM. Significance: Disrupted behavior, indicating neuronal damage in both sexes.	F: 0.3 mg/kg/day; M; 3.0 mg/kg/day	¹⁶

CNS, central nervous system; EPM, elevated plus maze; F, female; GABA, gamma-aminobutyric acid; GPR55, G-coupled protein receptor 55; hPBMECs, human primary brain microvascular endothelial cells; IL1β, interleukin 1β; IL6, interleukin-6; IL17, interleukin-17; iNOS, inducible nitric oxide synthase; L-DOPA, l-3,4-dihydroxyphenylalanine; LOEL, lowest-observed-effect level; M, male; MCA, middle cerebral artery; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PPARɣ, peroxisome proliferator-activated receptor gamma; PTZ, pentylenetetrazole; ROS, reactive oxygen species; Scn1a+/-, heterozygous loss of function SCN1A; SD, Sprague-Dawley rat; SH-SY5Y^APP+, SH-SY5Y cells transfected with the amyloid precursor protein; TNFα, tumor necrosis factor alpha; TSC, tuberous sclerosis complex; TRV1 or 2, transient receptor potential vanilloid type 1 or 2; USV, ultrasound vocalizations; VCAM1, vascular cell adhesion molecule-1. Table adapted from the review by Silvestro, Schepici, Bramanti, and Mazzon,¹⁸³ with permissions: Creative Commons — Attribution 4.0 International — CC BY 4.0.

In adults, aspects of CBD neurotoxicity are related to sex and strain in rodent studies.²⁰⁸ For example, male and female Swiss and C57BL/6 mice were treated with a single dose of CBD at 0 (saline/Tween 80), 10, and 20 mg/kg/day, and Flanders-sensitive line rats and Flanders- resistant line rats were treated with CBD at 0, 10, 30, and 60 mg/kg/day i.p. The mice were tested in the elevated plus maze, which measures anxiety behavior, and in the tail suspension test, which measures immobility and antidepressant behavior) 30 min after treatment. There were no effects from treatment with either strain of females in the tests, but male Swiss mice showed increased immobility in the tail suspension test at all doses (antidepressant). In the elevated plus maze test, the female Swiss mice showed decreased entries into the enclosed arm, indicating decreased exploratory behavior (antidepressant-like effect). Meanwhile, male and female C57BL/6 mice did not show effects in the elevated plus maze test. Rats were also tested 50 min after treatment in the forced swim and open field tests. The Flanders-sensitive line rats showed decreases at all doses in the forced swim test (measure of immobility), with no effects on distance traveled in the open field test and no effects in these tests with Flanders-resistant line rats. When the interval between treatment and testing was increased to 2 h, there was a slight increase in immobility in the Flanders-sensitive line rats at 30 mg/kg CBD. Therefore, it is significant to note that the exposure time, sex, strain, and species differences with CBD treatment were related to anxiety/depressive behaviors. The doses used in this study and those shown in Table 5 are within the range of those showing neuroprotection in Table 3, also administered i.p. In-vitro studies with mouse embryos also support the toxic effects of CBD during development.¹⁶

Animal studies have shown that doses of CBD that are neuroprotective (Table 3), can be toxic to the male reproductive tract.^{14,15,223–225} CBD treatment at 15 mg/kg/day (gavage) for three sperm development cycles in mice can lead to disrupted sperm development, abnormal seminiferous epithelium, decreased testes weights, and other effects that would impact fertility.¹⁴ Studies also have demonstrated reduced testosterone, inhibition of sperm maturation, and thinning, atrophied cells, pyknosis in seminiferous tubules, and other pathologies.¹⁴ The presumptive MOA involves CBD inhibition of 17α-hydroxylase in Leydig cells, leading to decreased testosterone production. However, in humans, the effects on sperm and other reproductive parameters in males have been mainly attributed to the ^Δ9THC content in cannabis, rather than CBD.^226,227 But based on animal studies, CBD in cannabis could contribute to the negative effects in males; hence, this area needs more research. In-vitro studies performed on human and mouse Sertoli cells obtained postnatally support the toxic effects of CBD observed in animal studies.¹⁸ Dose exposure, route, species, sex, frequency of consumption, and susceptibility to the effects from exposure contribute to health outcomes.

Future directions

With increasing use of cannabis with higher concentrations of ^Δ9THC, there are concomitant risks to safety in the general population from intoxication while driving or in the workplace. Methods have been developed to measure impairment from cannabis in a timely manner on site (e.g., in a car or workplace) through brain imaging to provide assessments of intoxication.³⁹ Functional near-infrared spectroscopy provides a measurable signature of neural impairment of the PFC, and the results are supported by blood and urine assessments to indicate whether participants were exposed but not impaired or exposed and impaired. Such measures acknowledge the growing need for detection and mitigating safety measures due to cognitive impairment from cannabis use.

Neurotoxicity of CBD is also in need of more study. For example, CBD injured neonatal rat cortical neurons and astrocytes in vitro at low therapeutic levels that could affect patients treated with CBD.^17,221 CBD is known to be neuroprotective in Parkinson’s disease, where dopaminergic neurons of the substantia nigra pars compacta are shown to degenerate.^78,194,229 Conversely, in animal models, dopaminergic pathways are attenuated by CBD, resulting in decreased motor functions.²⁴ While data indicate that for some, the benefits of CBD may outweigh the risks, it is clearly necessary to continue researching optimal treatment levels related to disease improvement. Persons exposed to higher doses of CBD for severe illnesses, such as DS to control seizures (Epidiolex®, Epidyolex® in Europe), may need to weigh the risk versus benefit and exert caution for use in pregnant women and children.

Finally, one of the biggest challenges in characterizing the effects of cannabis during developmental life stages is knowing the exposure and individual health risk factors. In laboratory experiments, the exact dose, purity of cannabinoids, animal strain/sex/pregnancy status, duration of exposure, and other parameters are controlled; however, with human subjects, it is difficult to characterize exposure. Nevertheless, knowledge of the dose and product components being consumed as well as the life stage of exposure, route of exposure (i.e., inhalation, s.c., i.v., oral, or i.p.), body fat composition, age, health status, frequency of use, and other factors will determine the absorption, distribution, and metabolism of cannabinoids entering the blood stream.^107,111 Many of these parameters are not consistent among studies performed in animals (e.g., different animal species/strains, dosing regimens, vehicles), and data may be difficult to obtain in epidemiological studies with human subjects. Thus, there is a need for further study to protect fetuses, infants, and children from harmful exposures during development. There is also a need for further research related to risks for male reproductive toxicity.

Conclusions

This review focused on neurotoxicity and neuroprotection of the most thoroughly characterized phytocannabinoids in cannabis—^Δ9THC and CBD. Most cannabis exposure is not a pure form of either compound, but it contains a combination of those and over 100 others. Due to the increasing use of cannabis or CBD, not just recreationally, but for the treatment of diseases (e.g., depression, anxiety, inflammation, pain, and seizures) and a plethora of other conditions, it is critical for the industry to thoroughly characterize expected exposures. The extent of the risk versus beneficial effects of compounds in cannabis is dependent on many factors, but, as indicated by studies with ^Δ9THC, there is a high risk for long-lasting neurodevelopmental effects from exposure to fetuses, infants, children, and adolescents, including severe mental dysfunction (e.g., depression, anxiety, and schizophrenia), decreased cognition, drug dependency tendencies, and decreased motor function. Adolescent use can present unique challenges because adolescence is a developmental stage of increased independence and potential for experimentation with cannabis. In addition, brain development as well as major dynamic changes in the eCBS continue for the first 25, or more, years of life; hence, cannabis exposure during adolescence can still attenuate brain development. Adolescent exposure has been shown to lead to persistent adverse neurodevelopmental changes, increasing the risks for major depressive disorder, drug addiction, and severe psychotic disorders.

On the other hand, CBD is nonpsychotropic and has positive therapeutic applications to treat childhood epilepsy, multiple sclerosis, stroke, Alzheimer’s disease, Parkinson’s disease, and other severe disorders. The focus has been mainly on the health benefits; however, the reported developmental effects from exposure in utero, effects on male reproduction, and associations with human genotoxicity have not been well studied, and a significant data gap remains.

Abbreviations

ACh:: acetylcholine

2-AG:: 2-arachidonoylglycerol

5-HT:: serotonin

AEA:: anandamide

ARfD:: acute reference dose

βA:: beta-amyloid

BDNF:: brain-derived neurotropic factor

Ca⁺²:: calcium

CB1R:: cannabinoid 1 receptor

CBD:: cannabidiol

CNS:: central nervous system

COX2:: cyclooxygenase-2

D1 or D2:: dopamine receptors

DA:: dopamine

DAGL:: diacylglycerol lipase

DRN:: dorsal raphe nucleus

DS:: Dravet syndrome

eCB:: endocannabinoid

eCBS:: endocannabinoid system

FAAH:: fatty acid amide hydrolase

GABA:: gamma-aminobutyric acid

GD:: gestation day

GPR55:: G-coupled protein receptor 55

i.p.:: intraperitoneal

i.v.:: intravenous

IL:: interleukin

iNOS:: inducible nitric oxide synthase

K⁺:: potassium

LOAEL:: lowest-observed-adverse-effect level

LOEL:: lowest-observed-effect level

MAGL:: monoacylglycerol lipase

MOA:: mode of action

NAc:: nucleus accumbens

NFкB:: nuclear factor kappa-light-chain-enhancer of activated B cells

NMDA:: N-methyl-D-aspartate

NOAEL:: no-observed-adverse-effect level

PFC:: prefrontal cortex

PPARɣ:: peroxisome proliferator-activated receptor gamma

ROS:: reactive oxygen species

s.c.:: subcutaneous

SNc:: substantia nigra

TNF:: tumor necrosis factor

TRPV1:: transient receptor potential cation channel subfamily V member 1, or vanilloid receptor 1

VTA:: ventral tegmental area

^Δ9THC:: delta-9-tetrahydrocannabinol

Declarations

Acknowledgement

I would like to thank Dr. Poorni Iyer, DVM, PhD for helpful discussions and for her work with cannabis and developmental effects.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

I have no competing interests (financial/personal) to declare.

Authors’ contributions

MHS is the sole author of this work.

References

1	Graupensperger S, Fleming CB, Jaffe AE, Rhew IC, Patrick ME, Lee CM. Changes in Young Adults’ Alcohol and Marijuana Use, Norms, and Motives from Before to During the COVID-19 Pandemic. J Adolesc Health 2021;68(4):658-665 View Article PubMed/NCBI

2	Coley RL, Hawkins SS, Ghiani M, Kruzik C, Baum CF. A quasi-experimental evaluation of marijuana policies and youth marijuana use. Am J Drug Alcohol Abuse 2019;45(3):292-303 View Article PubMed/NCBI

3	Hanuš LO, Meyer SM, Muñoz E, Taglialatela-Scafati O, Appendino G. Phytocannabinoids: a unified critical inventory. Nat Prod Rep 2016;33(12):1357-1392 View Article PubMed/NCBI

4	Pertwee RG, Cascio MG. Handbook of Cannabis. Oxford: Oxford Academic; 2015, 115-136 View Article

5	Schultes RE. Hallucinogens of plant origin. Science 1969;163(3864):245-254 View Article PubMed/NCBI

6	Raber JC, Elzinga S, Kaplan C. Understanding dabs: contamination concerns of cannabis concentrates and cannabinoid transfer during the act of dabbing. J Toxicol Sci 2015;40(6):797-803 View Article PubMed/NCBI

7	Russell C, Rueda S, Room R, Tyndall M, Fischer B. Routes of administration for cannabis use - basic prevalence and related health outcomes: A scoping review and synthesis. Int J Drug Policy 2018;52:87-96 View Article PubMed/NCBI

8	Musshoff F, Madea B. Review of biologic matrices (urine, blood, hair) as indicators of recent or ongoing cannabis use. Ther Drug Monit 2006;28(2):155-163 View Article PubMed/NCBI

9	Huestis MA. Handb Exp Pharmacol. ; 2005, 657-690 View Article PubMed/NCBI

10	European Food Safety Authority (EFSA). Scientific opinion on the risks for human health related to the presence of tetrahydrocannabinol (THC) in milk and other food of animal origin. EFSA Journal 2015;13(6):4141 View Article

11	Scheyer AF, Borsoi M, Wager-Miller J, Pelissier-Alicot AL, Murphy MN, Mackie K, et al. Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors. Biol Psychiatry 2020;87(7):666-677 View Article PubMed/NCBI

12	Pinkhasova DV, Jameson LE, Conrow KD, Simeone MP, Davis AP, Wiegers TC, et al. Regulatory Status of Pesticide Residues in Cannabis: Implications to Medical Use in Neurological Diseases. Curr Res Toxicol 2021;2:140-148 View Article PubMed/NCBI

Koppel BS, Brust JC, Fife T, Bronstein J, Youssof S, Gronseth G, et al. Systematic review: efficacy and safety of medical marijuana in selected neurologic disorders: report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology 2014;82(17):1556-1563 View Article PubMed/NCBI

14	Carvalho RK, Andersen ML, Mazaro-Costa R. The effects of cannabidiol on male reproductive system: A literature review. J Appl Toxicol 2020;40(1):132-150 View Article PubMed/NCBI

15	Carvalho RK, Rocha TL, Fernandes FH, Gonçalves BB, Souza MR, Araújo AA, et al. Decreasing sperm quality in mice subjected to chronic cannabidiol exposure: New insights of cannabidiol-mediated male reproductive toxicity. Chem Biol Interact 2022;351:109743 View Article PubMed/NCBI

16	Fabris D, Carvalho MC, Brandão ML, Prado WA, Zuardi AW, Crippa JA, et al. Sex-dependent differences in the anxiolytic-like effect of cannabidiol in the elevated plus-maze. J Psychopharmacol 2022;36(12):1371-1383 View Article PubMed/NCBI

17	Jurič DM, Bulc Rozman K, Lipnik-Štangelj M, Šuput D, Brvar M. Cytotoxic Effects of Cannabidiol on Neonatal Rat Cortical Neurons and Astrocytes: Potential Danger to Brain Development. Toxins (Basel) 2022;14(10):720 View Article PubMed/NCBI

18	Li Y, Wu Q, Li X, Von Tungeln LS, Beland FA, Petibone D, et al. In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells. Food Chem Toxicol 2022;159:112722 View Article PubMed/NCBI

19	Henschke P. Cannabis: An ancient friend or foe? What works and doesn’t work. Semin Fetal Neonatal Med 2019;24(2):149-154 View Article PubMed/NCBI

20	Jaques SC, Kingsbury A, Henshcke P, Chomchai C, Clews S, Falconer J, et al. Cannabis, the pregnant woman and her child: weeding out the myths. J Perinatol 2014;34(6):417-424 View Article PubMed/NCBI

21	Alpár A, Di Marzo V, Harkany T. At the Tip of an Iceberg: Prenatal Marijuana and Its Possible Relation to Neuropsychiatric Outcome in the Offspring. Biol Psychiatry 2016;79(7):e33-e45 View Article PubMed/NCBI

22	Monfort A, Ferreira E, Leclair G, Lodygensky GA. Pharmacokinetics of Cannabis and Its Derivatives in Animals and Humans During Pregnancy and Breastfeeding. Front Pharmacol 2022;13:919630 View Article PubMed/NCBI

23	Gheasuddin Y, Galea GL. Cannabidiol impairs neural tube closure in mouse whole embryo culture. Birth Defects Res 2022;114(18):1186-1193 View Article PubMed/NCBI

24	Iezzi D, Caceres-Rodriguez A, Chavis P, Manzoni OJJ. In utero exposure to cannabidiol disrupts select early-life behaviors in a sex-specific manner. Transl Psychiatry 2022;12(1):501 View Article PubMed/NCBI

25	ElSohly MA, Mehmedic Z, Foster S, Gon C, Chandra S, Church JC. Changes in Cannabis Potency Over the Last 2 Decades (1995-2014): Analysis of Current Data in the United States. Biol Psychiatry 2016;79(7):613-619 View Article PubMed/NCBI

26	Sarne Y, Asaf F, Fishbein M, Gafni M, Keren O. The dual neuroprotective-neurotoxic profile of cannabinoid drugs. Br J Pharmacol 2011;163(7):1391-1401 View Article PubMed/NCBI

27	Baglot SL, Hume C, Petrie GN, Aukema RJ, Lightfoot SHM, Grace LM, et al. Pharmacokinetics and central accumulation of delta-9-tetrahydrocannabinol (THC) and its bioactive metabolites are influenced by route of administration and sex in rats. Sci Rep 2021;11(1):23990 View Article PubMed/NCBI

28	Dow-Edwards D, Silva L. Endocannabinoids in brain plasticity: Cortical maturation, HPA axis function and behavior. Brain Res 2017;1654(Pt B):157-164 View Article PubMed/NCBI

29	Miller ML, Chadwick B, Dickstein DL, Purushothaman I, Egervari G, Rahman T, et al. Adolescent exposure to Δ(9)-tetrahydrocannabinol alters the transcriptional trajectory and dendritic architecture of prefrontal pyramidal neurons. Mol Psychiatry 2019;24(4):588-600 View Article PubMed/NCBI

30	Kong KL, Lee JK, Shisler S, Thanos PK, Huestis MA, Hawk L, et al. Prenatal tobacco and cannabis co-exposure and offspring obesity development from birth to mid-childhood. Pediatr Obes 2023;18(5):e13010 View Article PubMed/NCBI

31	Breijyeh Z, Jubeh B, Bufo SA, Karaman R, Scrano L. Cannabis: A Toxin-Producing Plant with Potential Therapeutic Uses. Toxins (Basel) 2021;13(2):117 View Article PubMed/NCBI

32	Chadwick B, Miller ML, Hurd YL. Cannabis Use during Adolescent Development: Susceptibility to Psychiatric Illness. Front Psychiatry 2013;4:129 View Article PubMed/NCBI

33	Bolhuis K, Kushner SA, Yalniz S, Hillegers MHJ, Jaddoe VWV, Tiemeier H, et al. Maternal and paternal cannabis use during pregnancy and the risk of psychotic-like experiences in the offspring. Schizophr Res 2018;202:322-327 View Article PubMed/NCBI

34	Fergusson DM, Horwood LJ, Northstone K, ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. Maternal use of cannabis and pregnancy outcome. BJOG 2002;109(1):21-27 View Article PubMed/NCBI

35	Gillies R, Lee K, Vanin S, Laviolette SR, Holloway AC, Arany E, et al. Maternal exposure to Δ9-tetrahydrocannabinol impairs female offspring glucose homeostasis and endocrine pancreatic development in the rat. Reprod Toxicol 2020;94:84-91 View Article PubMed/NCBI

36	Bouquet E, Eiden C, Fauconneau B, Pion C, Pain S, Pérault-Pochat MC, et al. Adverse events of recreational cannabis use during pregnancy reported to the French Addictovigilance Network between 2011 and 2020. Sci Rep 2022;12(1):16509 View Article PubMed/NCBI

37	Garry A, Rigourd V, Amirouche A, Fauroux V, Aubry S, Serreau R. Cannabis and breastfeeding. J Toxicol 2009;2009:596149 View Article PubMed/NCBI

38	Baker T, Datta P, Rewers-Felkins K, Thompson H, Kallem RR, Hale TW. Transfer of Inhaled Cannabis Into Human Breast Milk. Obstet Gynecol 2018;131(5):783-788 View Article PubMed/NCBI

39	Gilman JM, Schmitt WA, Potter K, Kendzior B, Pachas GN, Hickey S, et al. Identification of Δ9-tetrahydrocannabinol (THC) impairment using functional brain imaging. Neuropsychopharmacology 2022;47(4):944-952 View Article PubMed/NCBI

Deiana S, Watanabe A, Yamasaki Y, Amada N, Arthur M, Fleming S, et al. Plasma and brain pharmacokinetic profile of cannabidiol (CBD), cannabidivarine (CBDV), Δ⁹-tetrahydrocannabivarin (THCV) and cannabigerol (CBG) in rats and mice following oral and intraperitoneal administration and CBD action on obsessive-compulsive behaviour. Psychopharmacology (Berl) 2012;219(3):859-873 View Article PubMed/NCBI

41	Turner SE, Williams CM, Iversen L, Whalley BJ. Molecular Pharmacology of Phytocannabinoids. Prog Chem Org Nat Prod 2017;103:61-101 View Article PubMed/NCBI

42	Antonazzo M, Botta M, Bengoetxea H, Ruiz-Ortega JÁ, Morera-Herreras T. Therapeutic potential of cannabinoids as neuroprotective agents for damaged cells conducing to movement disorders. Int Rev Neurobiol 2019;146:229-257 View Article PubMed/NCBI

43	Cristino L, Bisogno T, Di Marzo V. Cannabinoids and the expanded endocannabinoid system in neurological disorders. Nat Rev Neurol 2020;16(1):9-29 View Article PubMed/NCBI

44	Ibeas Bih C, Chen T, Nunn AV, Bazelot M, Dallas M, Whalley BJ. Molecular Targets of Cannabidiol in Neurological Disorders. Neurotherapeutics 2015;12(4):699-730 View Article PubMed/NCBI

45	Brown JD, Winterstein AG. Potential Adverse Drug Events and Drug-Drug Interactions with Medical and Consumer Cannabidiol (CBD) Use. J Clin Med 2019;8(7):989 View Article PubMed/NCBI

46	Chesney E, McGuire P, Freeman TP, Strang J, Englund A. Lack of evidence for the effectiveness or safety of over-the-counter cannabidiol products. Ther Adv Psychopharmacol 2020;10:2045125320954992 View Article PubMed/NCBI

47	Di Marzo V. CB(1) receptor antagonism: biological basis for metabolic effects. Drug Discov Today 2008;13(23-24):1026-1041 View Article PubMed/NCBI

48	Mato S, Del Olmo E, Pazos A. Ontogenetic development of cannabinoid receptor expression and signal transduction functionality in the human brain. Eur J Neurosci 2003;17(9):1747-1754 View Article PubMed/NCBI

49	Zou S, Kumar U. Cannabinoid Receptors and the Endocannabinoid System: Signaling and Function in the Central Nervous System. Int J Mol Sci 2018;19(3):833 View Article PubMed/NCBI

50	Lu HC, Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry 2016;79(7):516-525 View Article PubMed/NCBI

51	Joshi N, Onaivi ES. Endocannabinoid System Components: Overview and Tissue Distribution. Adv Exp Med Biol 2019;1162:1-12 View Article PubMed/NCBI

52	Mir HD, Giorgini G, Di Marzo V. The emerging role of the endocannabinoidome-gut microbiome axis in eating disorders. Psychoneuroendocrinology 2023;154:106295 View Article PubMed/NCBI

53	Pertwee RG, Howlett AC, Abood ME, Alexander SP, Di Marzo V, Elphick MR, et al. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB₁ and CB₂. Pharmacol Rev 2010;62(4):588-631 View Article PubMed/NCBI

54	McAllister SD, Glass M. CB(1) and CB(2) receptor-mediated signalling: a focus on endocannabinoids. Prostaglandins Leukot Essent Fatty Acids 2002;66(2-3):161-171 View Article PubMed/NCBI

55	De Petrocellis L, Cascio MG, Di Marzo V. The endocannabinoid system: a general view and latest additions. Br J Pharmacol 2004;141(5):765-774 View Article PubMed/NCBI

56	Di Marzo V, Melck D, Bisogno T, De Petrocellis L. Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci 1998;21(12):521-528 View Article PubMed/NCBI

57	Di Marzo V, Piscitelli F, Mechoulam R. Handb Exp Pharmacol. ; 2011, 75-104 View Article PubMed/NCBI

58	Ahn K, McKinney MK, Cravatt BF. Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem Rev 2008;108(5):1687-1707 View Article PubMed/NCBI

59	Ibsen MS, Connor M, Glass M. Cannabinoid CB(1) and CB(2) Receptor Signaling and Bias. Cannabis Cannabinoid Res 2017;2(1):48-60 View Article PubMed/NCBI

60	Guo J, Ikeda SR. Endocannabinoids modulate N-type calcium channels and G-protein-coupled inwardly rectifying potassium channels via CB1 cannabinoid receptors heterologously expressed in mammalian neurons. Mol Pharmacol 2004;65(3):665-674 View Article PubMed/NCBI

61	Twitchell W, Brown S, Mackie K. Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat hippocampal neurons. J Neurophysiol 1997;78(1):43-50 View Article PubMed/NCBI

62	Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, et al. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002;54(2):161-202 View Article PubMed/NCBI

63	Fortin DA, Levine ES. Differential effects of endocannabinoids on glutamatergic and GABAergic inputs to layer 5 pyramidal neurons. Cereb Cortex 2007;17(1):163-174 View Article PubMed/NCBI

64	Karson MA, Whittington KC, Alger BE. Cholecystokinin inhibits endocannabinoid-sensitive hippocampal IPSPs and stimulates others. Neuropharmacology 2008;54(1):117-128 View Article PubMed/NCBI

65	Haj-Dahmane S, Shen RY. Regulation of plasticity of glutamate synapses by endocannabinoids and the cyclic-AMP/protein kinase A pathway in midbrain dopamine neurons. J Physiol 2010;588(Pt ;14):2589-2604 View Article PubMed/NCBI

66	Wang J, Shen RY, Haj-Dahmane S. Endocannabinoids mediate the glucocorticoid-induced inhibition of excitatory synaptic transmission to dorsal raphe serotonin neurons. J Physiol 2012;590(22):5795-5808 View Article PubMed/NCBI

67	Lau T, Schloss P. The cannabinoid CB1 receptor is expressed on serotonergic and dopaminergic neurons. Eur J Pharmacol 2008;578(2-3):137-141 View Article PubMed/NCBI

68	Laviolette SR, Grace AA. The roles of cannabinoid and dopamine receptor systems in neural emotional learning circuits: implications for schizophrenia and addiction. Cell Mol Life Sci 2006;63(14):1597-1613 View Article PubMed/NCBI

Kirilly E, Hunyady L, Bagdy G. Opposing local effects of endocannabinoids on the activity of noradrenergic neurons and release of noradrenaline: relevance for their role in depression and in the actions of CB(1) receptor antagonists. J Neural Transm (Vienna) 2013;120(1):177-186 View Article PubMed/NCBI

70	Mendiguren A, Aostri E, Pineda J. Regulation of noradrenergic and serotonergic systems by cannabinoids: relevance to cannabinoid-induced effects. Life Sci 2018;192:115-127 View Article PubMed/NCBI

71	Martin HG, Bernabeu A, Lassalle O, Bouille C, Beurrier C, Pelissier-Alicot AL, et al. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex. Front Cell Neurosci 2015;9:457 View Article PubMed/NCBI

72	Steffens M, Szabo B, Klar M, Rominger A, Zentner J, Feuerstein TJ. Modulation of electrically evoked acetylcholine release through cannabinoid CB1 receptors: evidence for an endocannabinoid tone in the human neocortex. Neuroscience 2003;120(2):455-465 View Article PubMed/NCBI

73	Berghuis P, Rajnicek AM, Morozov YM, Ross RA, Mulder J, Urbán GM, et al. Hardwiring the brain: endocannabinoids shape neuronal connectivity. Science 2007;316(5828):1212-1216 View Article PubMed/NCBI

74	Jiang R, Yamaori S, Takeda S, Yamamoto I, Watanabe K. Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes. Life Sci 2011;89(5-6):165-170 View Article PubMed/NCBI

75	Bernasconi C, Pelkonen O, Andersson TB, Strickland J, Wilk-Zasadna I, Asturiol D, et al. Validation of in vitro methods for human cytochrome P450 enzyme induction: Outcome of a multi-laboratory study. Toxicol In Vitro 2019;60:212-228 View Article PubMed/NCBI

76	Saili KS, Antonijevic T, Zurlinden TJ, Shah I, Deisenroth C, Knudsen TB. Molecular characterization of a toxicological tipping point during human stem cell differentiation. Reprod Toxicol 2020;91:1-13 View Article PubMed/NCBI

77	Mackie K. Handb Exp Pharmacol. ; 2005, 299-325 View Article PubMed/NCBI

78	Fernández-Ruiz J, Hernández M, Ramos JA. Cannabinoid-dopamine interaction in the pathophysiology and treatment of CNS disorders. CNS Neurosci Ther 2010;16(3):e72-e91 View Article PubMed/NCBI

79	Lupica CR, Riegel AC. Endocannabinoid release from midbrain dopamine neurons: a potential substrate for cannabinoid receptor antagonist treatment of addiction. Neuropharmacology 2005;48(8):1105-1116 View Article PubMed/NCBI

80	Peters KZ, Cheer JF, Tonini R. Modulating the Neuromodulators: Dopamine, Serotonin, and the Endocannabinoid System. Trends Neurosci 2021;44(6):464-477 View Article PubMed/NCBI

81	Ayano G. Dopamine: Receptors, Functions, Synthesis, Pathways, Locations and Mental Disorders: Review of Literatures. J Ment Disord Treat 2016;2(120):2 View Article

82	Alcaro A, Huber R, Panksepp J. Behavioral functions of the mesolimbic dopaminergic system: an affective neuroethological perspective. Brain Res Rev 2007;56(2):283-321 View Article PubMed/NCBI

83	Covey DP, Mateo Y, Sulzer D, Cheer JF, Lovinger DM. Endocannabinoid modulation of dopamine neurotransmission. Neuropharmacology 2017;124:52-61 View Article PubMed/NCBI

84	Liu Z, Lin R, Luo M. Reward Contributions to Serotonergic Functions. Annu Rev Neurosci 2020;43:141-162 View Article PubMed/NCBI

85	Haj-Dahmane S, Shen RY. Modulation of the serotonin system by endocannabinoid signaling. Neuropharmacology 2011;61(3):414-420 View Article PubMed/NCBI

86	Rodrigues da Silva N, Gomes FV, Sonego AB, Silva NRD, Guimarães FS. Cannabidiol attenuates behavioral changes in a rodent model of schizophrenia through 5-HT1A, but not CB1 and CB2 receptors. Pharmacol Res 2020;156:104749 View Article PubMed/NCBI

87	Yager LM, Garcia AF, Wunsch AM, Ferguson SM. The ins and outs of the striatum: role in drug addiction. Neuroscience 2015;301:529-541 View Article PubMed/NCBI

88	Ikemoto S. Brain reward circuitry beyond the mesolimbic dopamine system: a neurobiological theory. Neurosci Biobehav Rev 2010;35(2):129-150 View Article PubMed/NCBI

89	Melis M, Pistis M. Endocannabinoid signaling in midbrain dopamine neurons: more than physiology?. Curr Neuropharmacol 2007;5(4):268-277 View Article PubMed/NCBI

90	Morikawa H, Paladini CA. Dynamic regulation of midbrain dopamine neuron activity: intrinsic, synaptic, and plasticity mechanisms. Neuroscience 2011;198:95-111 View Article PubMed/NCBI

91	Peters KZ, Oleson EB, Cheer JF. A Brain on Cannabinoids: The Role of Dopamine Release in Reward Seeking and Addiction. Cold Spring Harb Perspect Med 2021;11(1):a039305 View Article PubMed/NCBI

92	Institute of Medicine (US) Forum on Neuroscience and Nervous System Disorders. Glutamate-Related Biomarkers in Drug Development for Disorders of the Nervous System: Workshop Summary. Washington (DC): National Academies Press (US); 2011 PubMed/NCBI

93	Zhang L, Wang M, Bisogno T, Di Marzo V, Alger BE. Endocannabinoids generated by Ca2+ or by metabotropic glutamate receptors appear to arise from different pools of diacylglycerol lipase. PLoS One 2011;6(1):e16305 View Article PubMed/NCBI

94	Berghuis P. Brain-derived neurotrophic factor and endocannabinoid functions in gabaergic interneuron development. Karolinska Instituet; 2007

95	Lee SH, Ledri M, Tóth B, Marchionni I, Henstridge CM, Dudok B, et al. Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release. J Neurosci 2015;35(27):10039-10057 View Article PubMed/NCBI

96	Musella A, Fresegna D, Rizzo FR, Gentile A, Bullitta S, De Vito F, et al. A novel crosstalk within the endocannabinoid system controls GABA transmission in the striatum. Sci Rep 2017;7(1):7363 View Article PubMed/NCBI

97	Barth C, Villringer A, Sacher J. Sex hormones affect neurotransmitters and shape the adult female brain during hormonal transition periods. Front Neurosci 2015;9:37 View Article PubMed/NCBI

98	Ogawa SK, Cohen JY, Hwang D, Uchida N, Watabe-Uchida M. Organization of monosynaptic inputs to the serotonin and dopamine neuromodulatory systems. Cell Rep 2014;8(4):1105-1118 View Article PubMed/NCBI

99	Leung MCK, Silva MH, Palumbo AJ, Lohstroh PN, Koshlukova SE, DuTeaux SB. Adverse outcome pathway of developmental neurotoxicity resulting from prenatal exposures to cannabis contaminated with organophosphate pesticide residues. Reprod Toxicol 2019;85:12-18 View Article PubMed/NCBI

100	Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, et al. Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 2007;10(7):870-879 View Article PubMed/NCBI

101

Campbell MA, Iyer P, Kaufman F, Kim A, Moran F, Niknam Y, et al. Animal evidence considered in determination of cannabis smoke and Δ(9) -tetrahydrocannabinol as causing reproductive toxicity (developmental endpoint); Part I. Somatic development. Birth Defects Res 2022;114(18):1143-1154 View Article PubMed/NCBI

102

Iyer P, Niknam Y, Campbell M, Moran F, Kaufman F, Kim A, et al. Animal evidence considered in determination of cannabis smoke and Δ(9) -tetrahydrocannabinol (Δ(9) -THC) as causing reproductive toxicity (developmental endpoint); Part II. Neurodevelopmental effects. Birth Defects Res 2022;114(18):1155-1168 View Article PubMed/NCBI

103	Iyer P, Watanabe M, Artinger KB. Emerging understanding of the effects of cannabis use during pregnancy. Birth Defects Res 2023;115(2):129-132 View Article PubMed/NCBI

104

OEHHA. Cannabis (Marijuana) Smoke. In: The Proposition 65 List, Office of Environmental Health Hazard Agency, California Environmental Protection Agency: Sacramento, California, 2020; Vol. January 3, 2020. Available from: https://oehha.ca.gov/proposition-65/proposition-65-list/. Accessed April 11, 2023

105	Meyer P, Langos M, Brenneisen R. Human Pharmacokinetics and Adverse Effects of Pulmonary and Intravenous THC-CBD Formulations. Med Cannabis Cannabinoids 2018;1(1):36-43 View Article PubMed/NCBI

106	Naef M, Russmann S, Petersen-Felix S, Brenneisen R. Development and pharmacokinetic characterization of pulmonal and intravenous delta-9-tetrahydrocannabinol (THC) in humans. J Pharm Sci 2004;93(5):1176-1184 View Article PubMed/NCBI

107

Hložek T, Uttl L, Kadeřábek L, Balíková M, Lhotková E, Horsley RR, et al. Pharmacokinetic and behavioural profile of THC, CBD, and THC+CBD combination after pulmonary, oral, and subcutaneous administration in rats and confirmation of conversion in vivo of CBD to THC. Eur Neuropsychopharmacol 2017;27(12):1223-1237 View Article PubMed/NCBI

108	Manwell LA, Mallet PE. Comparative effects of pulmonary and parenteral Δ⁹-tetrahydrocannabinol exposure on extinction of opiate-induced conditioned aversion in rats. Psychopharmacology (Berl) 2015;232(9):1655-1665 View Article PubMed/NCBI

109	Harkany T, Guzmán M, Galve-Roperh I, Berghuis P, Devi LA, Mackie K. The emerging functions of endocannabinoid signaling during CNS development. Trends Pharmacol Sci 2007;28(2):83-92 View Article PubMed/NCBI

110	Harkany T, Keimpema E, Barabás K, Mulder J. Endocannabinoid functions controlling neuronal specification during brain development. Mol Cell Endocrinol 2008;286(1-2 Suppl 1):S84-S90 View Article PubMed/NCBI

111

Manwell LA, Charchoglyan A, Brewer D, Matthews BA, Heipel H, Mallet PE. A vapourized Δ(9)-tetrahydrocannabinol (Δ(9)-THC) delivery system part I: development and validation of a pulmonary cannabinoid route of exposure for experimental pharmacology studies in rodents. J Pharmacol Toxicol Methods 2014;70(1):120-127 View Article PubMed/NCBI

112	DiNieri JA, Wang X, Szutorisz H, Spano SM, Kaur J, Casaccia P, et al. Maternal cannabis use alters ventral striatal dopamine D2 gene regulation in the offspring. Biol Psychiatry 2011;70(8):763-769 View Article PubMed/NCBI

113	Silva L, Zhao N, Popp S, Dow-Edwards D. Prenatal tetrahydrocannabinol (THC) alters cognitive function and amphetamine response from weaning to adulthood in the rat. Neurotoxicol Teratol 2012;34(1):63-71 View Article PubMed/NCBI

114	Spano MS, Ellgren M, Wang X, Hurd YL. Prenatal cannabis exposure increases heroin seeking with allostatic changes in limbic enkephalin systems in adulthood. Biol Psychiatry 2007;61(4):554-563 View Article PubMed/NCBI

115	Beggiato S, Borelli AC, Tomasini MC, Morgano L, Antonelli T, Tanganelli S, et al. Long-lasting alterations of hippocampal GABAergic neurotransmission in adult rats following perinatal Δ(9)-THC exposure. Neurobiol Learn Mem 2017;139:135-143 View Article PubMed/NCBI

116	Slotkin TA, Skavicus S, Levin ED, Seidler FJ. Paternal Δ9-Tetrahydrocannabinol Exposure Prior to Mating Elicits Deficits in Cholinergic Synaptic Function in the Offspring. Toxicol Sci 2020;174(2):210-217 View Article PubMed/NCBI

117

Watson CT, Szutorisz H, Garg P, Martin Q, Landry JA, Sharp AJ, et al. Genome-Wide DNA Methylation Profiling Reveals Epigenetic Changes in the Rat Nucleus Accumbens Associated With Cross-Generational Effects of Adolescent THC Exposure. Neuropsychopharmacology 2015;40(13):2993-3005 View Article PubMed/NCBI

118	Levin ED, Hawkey AB, Hall BJ, Cauley M, Slade S, Yazdani E, et al. Paternal THC exposure in rats causes long-lasting neurobehavioral effects in the offspring. Neurotoxicol Teratol 2019;74:106806 View Article PubMed/NCBI

119	Beiersdorf J, Hevesi Z, Calvigioni D, Pyszkowski J, Romanov R, Szodorai E, et al. Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development. JCI Insight 2020;5(23):135418 View Article PubMed/NCBI

120

Quinn HR, Matsumoto I, Callaghan PD, Long LE, Arnold JC, Gunasekaran N, et al. Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual cognitive deficits and changes in hippocampal protein expression following exposure. Neuropsychopharmacology 2008;33(5):1113-1126 View Article PubMed/NCBI

121

Murphy M, Mills S, Winstone J, Leishman E, Wager-Miller J, Bradshaw H, et al. Chronic Adolescent Δ(9)-Tetrahydrocannabinol Treatment of Male Mice Leads to Long-Term Cognitive and Behavioral Dysfunction, Which Are Prevented by Concurrent Cannabidiol Treatment. Cannabis Cannabinoid Res 2017;2(1):235-246 View Article PubMed/NCBI

122	Rubino T, Realini N, Braida D, Guidi S, Capurro V, Viganò D, et al. Changes in hippocampal morphology and neuroplasticity induced by adolescent THC treatment are associated with cognitive impairment in adulthood. Hippocampus 2009;19(8):763-772 View Article PubMed/NCBI

123	Winsauer PJ, Daniel JM, Filipeanu CM, Leonard ST, Hulst JL, Rodgers SP, et al. Long-term behavioral and pharmacodynamic effects of delta-9-tetrahydrocannabinol in female rats depend on ovarian hormone status. Addict Biol 2011;16(1):64-81 View Article PubMed/NCBI

124	Egerton A, Brett RR, Pratt JA. Acute delta9-tetrahydrocannabinol-induced deficits in reversal learning: neural correlates of affective inflexibility. Neuropsychopharmacology 2005;30(10):1895-1905 View Article PubMed/NCBI

125	Liu J. Effects of Cannabidiol and Δ 9-Tetrahydrocannabinol in the Elevated Plus Maze and Forced Swim Tests. Toronto: University of Toronto; 2019

126	Long LE, Chesworth R, Huang XF, McGregor IS, Arnold JC, Karl T. A behavioural comparison of acute and chronic Delta9-tetrahydrocannabinol and cannabidiol in C57BL/6JArc mice. Int J Neuropsychopharmacol 2010;13(7):861-876 View Article PubMed/NCBI

127	Hampson RE, Deadwyler SA. Cannabinoids reveal the necessity of hippocampal neural encoding for short-term memory in rats. J Neurosci 2000;20(23):8932-8942 View Article PubMed/NCBI

128	El-Alfy AT, Ivey K, Robinson K, Ahmed S, Radwan M, Slade D, et al. Antidepressant-like effect of delta9-tetrahydrocannabinol and other cannabinoids isolated from Cannabis sativa L. Pharmacol Biochem Behav. 2010;95(4):434-442 View Article PubMed/NCBI

129	Klein C, Karanges E, Spiro A, Wong A, Spencer J, Huynh T, et al. Cannabidiol potentiates Δ⁹-tetrahydrocannabinol (THC) behavioural effects and alters THC pharmacokinetics during acute and chronic treatment in adolescent rats. Psychopharmacology (Berl) 2011;218(2):443-457 View Article PubMed/NCBI

130	Szutorisz H, Egervári G, Sperry J, Carter JM, Hurd YL. Cross-generational THC exposure alters the developmental sensitivity of ventral and dorsal striatal gene expression in male and female offspring. Neurotoxicol Teratol 2016;58:107-114 View Article PubMed/NCBI

131	Bonnin A, de Miguel R, Castro JG, Ramos JA, Fernandez-Ruiz JJ. Effects of perinatal exposure to delta 9-tetrahydrocannabinol on the fetal and early postnatal development of tyrosine hydroxylase-containing neurons in rat brain. J Mol Neurosci 1996;7(4):291-308 View Article PubMed/NCBI

132

Sarikahya MH, Cousineau S, De Felice M, Lee K, Wong KK, DeVuono MV, et al. Prenatal THC Exposure Induces Sex-Dependent Neuropsychiatric Endophenotypes in Offspring and Long-Term Disruptions in Fatty-Acid Signaling Pathways Directly in the Mesolimbic Circuitry. ENEURO 2022;9(5):ENEURO.0253-22.2022 View Article PubMed/NCBI

133	Traccis F, Serra V, Sagheddu C, Congiu M, Saba P, Giua G, et al. Prenatal THC Does Not Affect Female Mesolimbic Dopaminergic System in Preadolescent Rats. Int J Mol Sci 2021;22(4):1666 View Article PubMed/NCBI

134	Sagheddu C, Traccis F, Serra V, Congiu M, Frau R, Cheer JF, et al. Mesolimbic dopamine dysregulation as a signature of information processing deficits imposed by prenatal THC exposure. Prog Neuropsychopharmacol Biol Psychiatry 2021;105:110128 View Article PubMed/NCBI

135

de Salas-Quiroga A, Díaz-Alonso J, García-Rincón D, Remmers F, Vega D, Gómez-Cañas M, et al. Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons. Proc Natl Acad Sci U S A 2015;112(44):13693-13698 View Article PubMed/NCBI

136	Tortoriello G, Morris CV, Alpar A, Fuzik J, Shirran SL, Calvigioni D, et al. Miswiring the brain: Δ9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway. EMBO J 2014;33(7):668-685 View Article PubMed/NCBI

137	Mohammed A, Alghetaa HK, Zhou J, Chatterjee S, Nagarkatti P, Nagarkatti M. Protective effects of Δ(9) -tetrahydrocannabinol against enterotoxin-induced acute respiratory distress syndrome are mediated by modulation of microbiota. Br J Pharmacol 2020;177(22):5078-5095 View Article PubMed/NCBI

138	Keeley R, Himmler S, Pellis S, McDonald R. Chronic exposure to Δ (9)-tetrahydrocannabinol in adolescence decreases social play behaviours. F1000Res 2021;10:1191 View Article PubMed/NCBI

139	Mallet PE, Beninger RJ. The cannabinoid CB1 receptor antagonist SR141716A attenuates the memory impairment produced by delta9-tetrahydrocannabinol or anandamide. Psychopharmacology (Berl) 1998;140(1):11-19 View Article PubMed/NCBI

140	Verrico CD, Jentsch JD, Roth RH, Taylor JR. Repeated, intermittent delta(9)-tetrahydrocannabinol administration to rats impairs acquisition and performance of a test of visuospatial divided attention. Neuropsychopharmacology 2004;29(3):522-529 View Article PubMed/NCBI

141

Jentsch JD, Andrusiak E, Tran A, Bowers MB, Roth RH. Delta 9-tetrahydrocannabinol increases prefrontal cortical catecholaminergic utilization and impairs spatial working memory in the rat: blockade of dopaminergic effects with HA966. Neuropsychopharmacology 1997;16(6):426-432 View Article PubMed/NCBI

142	Grant KS, Petroff R, Isoherranen N, Stella N, Burbacher TM. Cannabis use during pregnancy: Pharmacokinetics and effects on child development. Pharmacol Ther 2018;182:133-151 View Article PubMed/NCBI

143	Hurd YL, Manzoni OJ, Pletnikov MV, Lee FS, Bhattacharyya S, Melis M. Cannabis and the Developing Brain: Insights into Its Long-Lasting Effects. J Neurosci 2019;39(42):8250-8258 View Article PubMed/NCBI

144	Fried PA, Smith AM. A literature review of the consequences of prenatal marihuana exposure. An emerging theme of a deficiency in aspects of executive function. Neurotoxicol Teratol 2001;23(1):1-11 View Article PubMed/NCBI

145	Gunn JK, Rosales CB, Center KE, Nuñez A, Gibson SJ, Christ C, et al. Prenatal exposure to cannabis and maternal and child health outcomes: a systematic review and meta-analysis. BMJ Open 2016;6(4):e009986 View Article PubMed/NCBI

146	Jutras-Aswad D, DiNieri JA, Harkany T, Hurd YL. Neurobiological consequences of maternal cannabis on human fetal development and its neuropsychiatric outcome. Eur Arch Psychiatry Clin Neurosci 2009;259(7):395-412 View Article PubMed/NCBI

147	Richardson GA, Day NL, Goldschmidt L. Prenatal alcohol, marijuana, and tobacco use: infant mental and motor development. Neurotoxicol Teratol 1995;17(4):479-487 View Article PubMed/NCBI

148	Ramírez S, Miguez G, Quezada-Scholz VE, Pardo L, Alfaro F, Varas FI, et al. Behavioral effects on the offspring of rodent mothers exposed to Tetrahydrocannabinol (THC): A meta-analysis. Front Psychol 2022;13:934600 View Article PubMed/NCBI

149	Mohammed AN, Alugubelly N, Kaplan BL, Carr RL. Effect of repeated juvenile exposure to Δ9-tetrahydrocannabinol on anxiety-related behavior and social interactions in adolescent rats. Neurotoxicol Teratol 2018;69:11-20 View Article PubMed/NCBI

150	Metz TD, Stickrath EH. Marijuana use in pregnancy and lactation: a review of the evidence. Am J Obstet Gynecol 2015;213(6):761-778 View Article PubMed/NCBI

151	Kaila K, Price TJ, Payne JA, Puskarjov M, Voipio J. Cation-chloride cotransporters in neuronal development, plasticity and disease. Nat Rev Neurosci 2014;15(10):637-654 View Article PubMed/NCBI

152	Farrelly AM, Vlachou S. Effects of Cannabinoid Exposure during Neurodevelopment on Future Effects of Drugs of Abuse: A Preclinical Perspective. Int J Mol Sci 2021;22(18):9989 View Article PubMed/NCBI

153	Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev 2011;32(1):81-151 View Article PubMed/NCBI

154

Rubino T, Vigano’ D, Realini N, Guidali C, Braida D, Capurro V, et al. Chronic delta 9-tetrahydrocannabinol during adolescence provokes sex-dependent changes in the emotional profile in adult rats: behavioral and biochemical correlates. Neuropsychopharmacology 2008;33(11):2760-2771 View Article PubMed/NCBI

155	Karlsgodt KH, Sun D, Cannon TD. Structural and Functional Brain Abnormalities in Schizophrenia. Curr Dir Psychol Sci 2010;19(4):226-231 View Article PubMed/NCBI

156	Meyer HC, Lee FS, Gee DG. The Role of the Endocannabinoid System and Genetic Variation in Adolescent Brain Development. Neuropsychopharmacology 2018;43(1):21-33 View Article PubMed/NCBI

157	Curran HV, Freeman TP, Mokrysz C, Lewis DA, Morgan CJ, Parsons LH. Keep off the grass? Cannabis, cognition and addiction. Nat Rev Neurosci 2016;17(5):293-306 View Article PubMed/NCBI

158	Trezza V, Vanderschuren LJ. Bidirectional cannabinoid modulation of social behavior in adolescent rats. Psychopharmacology (Berl) 2008;197(2):217-227 View Article PubMed/NCBI

159	Iyengar U, Snowden N, Asarnow JR, Moran P, Tranah T, Ougrin D. A Further Look at Therapeutic Interventions for Suicide Attempts and Self-Harm in Adolescents: An Updated Systematic Review of Randomized Controlled Trials. Front Psychiatry 2018;9:583 View Article PubMed/NCBI

160	Miech R, Johnston L, O’Malley PM, Bachman JG, Patrick ME. Trends in Adolescent Vaping, 2017-2019. N Engl J Med 2019;381(15):1490-1491 View Article PubMed/NCBI

161	Trivers KF, Phillips E, Gentzke AS, Tynan MA, Neff LJ. Prevalence of Cannabis Use in Electronic Cigarettes Among US Youth. JAMA Pediatr 2018;172(11):1097-1099 View Article PubMed/NCBI

162	Silva MH. Chlorpyrifos and Δ(9) Tetrahydrocannabinol exposure and effects on parameters associated with the endocannabinoid system and risk factors for obesity. Curr Res Toxicol 2021;2:296-308 View Article PubMed/NCBI

163	Di Forti M, Sallis H, Allegri F, Trotta A, Ferraro L, Stilo SA, et al. Daily use, especially of high-potency cannabis, drives the earlier onset of psychosis in cannabis users. Schizophr Bull 2014;40(6):1509-1517 View Article PubMed/NCBI

164	Marconi A, Di Forti M, Lewis CM, Murray RM, Vassos E. Meta-analysis of the Association Between the Level of Cannabis Use and Risk of Psychosis. Schizophr Bull 2016;42(5):1262-1269 View Article PubMed/NCBI

165	Subodh BN, Sahoo S, Basu D, Mattoo SK. Age of onset of substance use in patients with dual diagnosis and its association with clinical characteristics, risk behaviors, course, and outcome: A retrospective study. Indian J Psychiatry 2019;61(4):359-368 View Article PubMed/NCBI

166	Agrawal A, Neale MC, Prescott CA, Kendler KS. A twin study of early cannabis use and subsequent use and abuse/dependence of other illicit drugs. Psychol Med 2004;34(7):1227-1237 View Article PubMed/NCBI

167	Pushkin AN, Eugene AJ, Lallai V, Torres-Mendoza A, Fowler JP, Chen E, et al. Cannabinoid and nicotine exposure during adolescence induces sex-specific effects on anxiety- and reward-related behaviors during adulthood. PLoS One 2019;14(1):e0211346 View Article PubMed/NCBI

168	Memedovich KA, Dowsett LE, Spackman E, Noseworthy T, Clement F. The adverse health effects and harms related to marijuana use: an overview review. CMAJ Open 2018;6(3):E339-E346 View Article PubMed/NCBI

169	Roberts VHJ, Schabel MC, Boniface ER, D’Mello RJ, Morgan TK, Terrobias JJD, et al. Chronic prenatal delta-9-tetrahydrocannabinol exposure adversely impacts placental function and development in a rhesus macaque model. Sci Rep 2022;12(1):20260 View Article PubMed/NCBI

170	Hedges JC, Hanna CB, Bash JC, Boniface ER, Burch FC, Mahalingaiah S, et al. Chronic exposure to delta-9-tetrahydrocannabinol impacts testicular volume and male reproductive health in rhesus macaques. Fertil Steril 2022;117(4):698-707 View Article PubMed/NCBI

171	Pinilla L, Aguilar E, Dieguez C, Millar RP, Tena-Sempere M. Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Rev 2012;92(3):1235-1316 View Article PubMed/NCBI

172	Ryan KS, Mahalingaiah S, Campbell LR, Roberts VHJ, Terrobias JJD, Naito CS, et al. The effects of delta-9-tetrahydrocannabinol exposure on female menstrual cyclicity and reproductive health in rhesus macaques. F S Sci 2021;2(3):287-294 View Article PubMed/NCBI

173	Frau R, Melis M. Sex-specific susceptibility to psychotic-like states provoked by prenatal THC exposure: Reversal by pregnenolone. J Neuroendocrinol 2023;35(2):e13240 View Article PubMed/NCBI

174	Marchei E, Escuder D, Pallas CR, Garcia-Algar O, Gómez A, Friguls B, et al. Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast milk by liquid chromatography tandem mass spectrometry. J Pharm Biomed Anal 2011;55(2):309-316 View Article PubMed/NCBI

175	Moore BF, Sauder KA, Shapiro ALB, Crume T, Kinney GL, Dabelea D. Fetal Exposure to Cannabis and Childhood Metabolic Outcomes: The Healthy Start Study. J Clin Endocrinol Metab 2022;107(7):e2862-e2869 View Article PubMed/NCBI

176	Arcella D, Cascio C, Mackay K, European Food Safety Authority (EFSA). Acute human exposure assessment to tetrahydrocannabinol (Δ(9)-THC). EFSA J 2020;18(1):e05953 View Article PubMed/NCBI

177	US EPA. A Review of the Reference Dose and Reference Concentration Processes. Available from: https://www.epa.gov/sites/production/files/2014-12/documents/rfd-final.pdf. Accessed April 11, 2023

178	WHO. Harmonization Project Document 11: Guidance Document on Evaluating and Expressing Uncertainty in Hazard Characterization. Available from: https://www.who.int/ipcs/methods/harmonization/uncertainty_in_hazard_characterization.pdf. Accessed April 11, 2023

179	World Health Organization & International Programme on Chemical Safety. Guidance document on evaluating and expressing uncertainty in hazard characterization. 2nd ed. 2018; xxii, p. 159. ISBN 9789241513548. Available from: https://apps.who.int/iris/handle/10665/259858. Accessed April 11, 2023

180	Maroon J, Bost J. Review of the neurological benefits of phytocannabinoids. Surg Neurol Int 2018;9:91 View Article PubMed/NCBI

181	Patricio F, Morales-Andrade AA, Patricio-Martínez A, Limón ID. Cannabidiol as a Therapeutic Target: Evidence of its Neuroprotective and Neuromodulatory Function in Parkinson’s Disease. Front Pharmacol 2020;11:595635 View Article PubMed/NCBI

182	Peres FF, Lima AC, Hallak JEC, Crippa JA, Silva RH, Abílio VC. Cannabidiol as a Promising Strategy to Treat and Prevent Movement Disorders?. Front Pharmacol 2018;9:482 View Article PubMed/NCBI

183	Silvestro S, Schepici G, Bramanti P, Mazzon E. Molecular Targets of Cannabidiol in Experimental Models of Neurological Disease. Molecules 2020;25(21):5186 View Article PubMed/NCBI

184	Gabbouj S, Ryhänen S, Marttinen M, Wittrahm R, Takalo M, Kemppainen S, et al. Altered Insulin Signaling in Alzheimer’s Disease Brain - Special Emphasis on PI3K-Akt Pathway. Front Neurosci 2019;13:629 View Article PubMed/NCBI

185	Werkman IL, Lentferink DH, Baron W. Macroglial diversity: white and grey areas and relevance to remyelination. Cell Mol Life Sci 2021;78(1):143-171 View Article PubMed/NCBI

186	Carrier EJ, Auchampach JA, Hillard CJ. Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc Natl Acad Sci U S A 2006;103(20):7895-7900 View Article PubMed/NCBI

187	Pandolfo P, Silveirinha V, dos Santos-Rodrigues A, Venance L, Ledent C, Takahashi RN, et al. Cannabinoids inhibit the synaptic uptake of adenosine and dopamine in the rat and mouse striatum. Eur J Pharmacol 2011;655(1-3):38-45 View Article PubMed/NCBI

188	Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of cannabidiol at 5-HT1a receptors. Neurochem Res 2005;30(8):1037-1043 View Article PubMed/NCBI

189	Lee JLC, Bertoglio LJ, Guimarães FS, Stevenson CW. Cannabidiol regulation of emotion and emotional memory processing: relevance for treating anxiety-related and substance abuse disorders. Br J Pharmacol 2017;174(19):3242-3256 View Article PubMed/NCBI

190	Marinho AL, Vila-Verde C, Fogaça MV, Guimarães FS. Effects of intra-infralimbic prefrontal cortex injections of cannabidiol in the modulation of emotional behaviors in rats: contribution of 5HT₁A receptors and stressful experiences. Behav Brain Res 2015;286:49-56 View Article PubMed/NCBI

191	Silvestri C, Pagano E, Lacroix S, Venneri T, Cristiano C, Calignano A, et al. Fish Oil, Cannabidiol and the Gut Microbiota: An Investigation in a Murine Model of Colitis. Front Pharmacol 2020;11:585096 View Article PubMed/NCBI

192	Britch SC, Babalonis S, Walsh SL. Cannabidiol: pharmacology and therapeutic targets. Psychopharmacology (Berl) 2021;238(1):9-28 View Article PubMed/NCBI

193	De Gregorio D, McLaughlin RJ, Posa L, Ochoa-Sanchez R, Enns J, Lopez-Canul M, et al. Cannabidiol modulates serotonergic transmission and reverses both allodynia and anxiety-like behavior in a model of neuropathic pain. Pain 2019;160(1):136-150 View Article PubMed/NCBI

194	Fernández-Ruiz J, Sagredo O, Pazos MR, García C, Pertwee R, Mechoulam R, et al. Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid?. Br J Clin Pharmacol 2013;75(2):323-333 View Article PubMed/NCBI

195	Gomes FV, Del Bel EA, Guimarães FS. Cannabidiol attenuates catalepsy induced by distinct pharmacological mechanisms via 5-HT1A receptor activation in mice. Prog Neuropsychopharmacol Biol Psychiatry 2013;46:43-47 View Article PubMed/NCBI

196	Mendiguren A, Aostri E, Alberdi E, Pérez-Samartín A, Pineda J. Functional characterization of cannabidiol effect on the serotonergic neurons of the dorsal raphe nucleus in rat brain slices. Front Pharmacol 2022;13:956886 View Article PubMed/NCBI

197	Chen Y, McCarron RM, Ohara Y, Bembry J, Azzam N, Lenz FA, et al. Human brain capillary endothelium: 2-arachidonoglycerol (endocannabinoid) interacts with endothelin-1. Circ Res 2000;87(4):323-327 View Article PubMed/NCBI

198	O’Sullivan SE. An update on PPAR activation by cannabinoids. Br J Pharmacol 2016;173(12):1899-1910 View Article PubMed/NCBI

199	Atalay S, Jarocka-Karpowicz I, Skrzydlewska E. Antioxidative and Anti-Inflammatory Properties of Cannabidiol. Antioxidants (Basel) 2019;9(1):21 View Article PubMed/NCBI

200	De Petrocellis L, Ligresti A, Moriello AS, Allarà M, Bisogno T, Petrosino S, et al. Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes. Br J Pharmacol 2011;163(7):1479-1494 View Article PubMed/NCBI

201	Howlett AC. Handb Exp Pharmacol. ; 2005, 53-79 View Article PubMed/NCBI

202	Leweke FM, Piomelli D, Pahlisch F, Muhl D, Gerth CW, Hoyer C, et al. Cannabidiol enhances anandamide signaling and alleviates psychotic symptoms of schizophrenia. Transl Psychiatry 2012;2(3):e94 View Article PubMed/NCBI

203	Marichal-Cancino BA, Fajardo-Valdez A, Ruiz-Contreras AE, Mendez-Díaz M, Prospero-García O. Advances in the Physiology of GPR55 in the Central Nervous System. Curr Neuropharmacol 2017;15(5):771-778 View Article PubMed/NCBI

204	Silvestro S, Mammana S, Cavalli E, Bramanti P, Mazzon E. Use of Cannabidiol in the Treatment of Epilepsy: Efficacy and Security in Clinical Trials. Molecules 2019;24(8):1459 View Article PubMed/NCBI

205	Cifelli P, Ruffolo G, De Felice E, Alfano V, van Vliet EA, Aronica E, et al. Phytocannabinoids in Neurological Diseases: Could They Restore a Physiological GABAergic Transmission?. Int J Mol Sci 2020;21(3):723 View Article PubMed/NCBI

206	Kaplan JS, Stella N, Catterall WA, Westenbroek RE. Cannabidiol attenuates seizures and social deficits in a mouse model of Dravet syndrome. Proc Natl Acad Sci U S A 2017;114(42):11229-11234 View Article PubMed/NCBI

207	Bakas T, van Nieuwenhuijzen PS, Devenish SO, McGregor IS, Arnold JC, Chebib M. The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABA(A) receptors. Pharmacol Res 2017;119:358-370 View Article PubMed/NCBI

208	Silote GP, Gatto MC, Eskelund A, Guimarães FS, Wegener G, Joca SRL. Strain-, Sex-, and Time-Dependent Antidepressant-like Effects of Cannabidiol. Pharmaceuticals (Basel) 2021;14(12):1269 View Article PubMed/NCBI

209	Kwan Cheung KA, Mitchell MD, Heussler HS. Cannabidiol and Neurodevelopmental Disorders in Children. Front Psychiatry 2021;12:643442 View Article PubMed/NCBI

210	Ożarowski M, Karpiński TM, Zielińska A, Souto EB, Wielgus K. Cannabidiol in Neurological and Neoplastic Diseases: Latest Developments on the Molecular Mechanism of Action. Int J Mol Sci 2021;22(9):4294 View Article PubMed/NCBI

211	Rubin R. The Path to the First FDA-Approved Cannabis-Derived Treatment and What Comes Next. JAMA 2018;320(12):1227-1229 View Article PubMed/NCBI

212	Blessing EM, Steenkamp MM, Manzanares J, Marmar CR. Cannabidiol as a Potential Treatment for Anxiety Disorders. Neurotherapeutics 2015;12(4):825-836 View Article PubMed/NCBI

213	Yarar E. Role and Function of Endocannabinoid System in Major Depressive Disease. Med Cannabis Cannabinoids 2021;4(1):1-12 View Article PubMed/NCBI

214	McGuire P, Robson P, Cubala WJ, Vasile D, Morrison PD, Barron R, et al. Cannabidiol (CBD) as an Adjunctive Therapy in Schizophrenia: A Multicenter Randomized Controlled Trial. Am J Psychiatry 2018;175(3):225-231 View Article PubMed/NCBI

215

Aguilera-Portillo G, Rangel-López E, Villeda-Hernández J, Chavarría A, Castellanos P, Elmazoglu Z, et al. The Pharmacological Inhibition of Fatty Acid Amide Hydrolase Prevents Excitotoxic Damage in the Rat Striatum: Possible Involvement of CB1 Receptors Regulation. Mol Neurobiol 2019;56(2):844-856 View Article PubMed/NCBI

216	Di Marzo V. Anandamide serves two masters in the brain. Nat Neurosci 2010;13(12):1446-1448 View Article PubMed/NCBI

217	Javed H, Azimullah S, Haque ME, Ojha SK. Cannabinoid Type 2 (CB2) Receptors Activation Protects against Oxidative Stress and Neuroinflammation Associated Dopaminergic Neurodegeneration in Rotenone Model of Parkinson’s Disease. Front Neurosci 2016;10:321 View Article PubMed/NCBI

218	Junior NCF, Dos-Santos-Pereira M, Guimarães FS, Del Bel E. Cannabidiol and Cannabinoid Compounds as Potential Strategies for Treating Parkinson’s Disease and L-DOPA-Induced Dyskinesia. Neurotox Res 2020;37(1):12-29 View Article PubMed/NCBI

219	Navarro G, Morales P, Rodríguez-Cueto C, Fernández-Ruiz J, Jagerovic N, Franco R. Targeting Cannabinoid CB2 Receptors in the Central Nervous System. Medicinal Chemistry Approaches with Focus on Neurodegenerative Disorders. Front Neurosci 2016;10:406 View Article PubMed/NCBI

220	Wilton DK, Stevens B. The contribution of glial cells to Huntington’s disease pathogenesis. Neurobiol Dis 2020;143:104963 View Article PubMed/NCBI

221	Millar SA, Stone NL, Yates AS, O’Sullivan SE. A Systematic Review on the Pharmacokinetics of Cannabidiol in Humans. Front Pharmacol 2018;9:1365 View Article PubMed/NCBI

222	Izzo AA, Borrelli F, Capasso R, Di Marzo V, Mechoulam R. Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacol Sci 2009;30(10):515-527 View Article PubMed/NCBI

223	Carvalho RK, Santos ML, Souza MR, Rocha TL, Guimarães FS, Anselmo-Franci JA, et al. Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice. J Appl Toxicol 2018;38(12):1545 View Article PubMed/NCBI

224	Carvalho RK, Santos ML, Souza MR, Rocha TL, Guimarães FS, Anselmo-Franci JA, et al. Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice. J Appl Toxicol 2018;38(9):1215-1223 View Article PubMed/NCBI

225	Carvalho RK, Souza MR, Santos ML, Guimarães FS, Pobbe RLH, Andersen ML, et al. Chronic cannabidiol exposure promotes functional impairment in sexual behavior and fertility of male mice. Reprod Toxicol 2018;81:34-40 View Article PubMed/NCBI

226	Reece AS, Hulse GK. Impacts of cannabinoid epigenetics on human development: reflections on Murphy et. al. ‘cannabinoid exposure and altered DNA methylation in rat and human sperm’ epigenetics 2018; 13: 1208-1221. Epigenetics 2019;14(11):1041-1056 View Article PubMed/NCBI

227	Reece AS, Hulse GK. Geotemporospatial and causal inference epidemiological analysis of US survey and overview of cannabis, cannabidiol and cannabinoid genotoxicity in relation to congenital anomalies 2001-2015. BMC Pediatr 2022;22(1):47 View Article PubMed/NCBI

228	Li Y, Li X, Cournoyer P, Choudhuri S, Yu X, Guo L, et al. Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells. Toxicol Sci 2023;191(2):227-238 View Article PubMed/NCBI

229	Thomas B, Beal MF. Parkinson’s disease. Hum Mol Genet 2007;16(R2):R183-R194 View Article PubMed/NCBI

Copyright © 2023 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neurotoxic or Protective Cannabis Components: Delta-9-Tetrahydrocannabinol (Δ9THC) and Cannabidiol (CBD)