**4. Studied vertebrate**

#### **4.1 Inside the ECS**

This section will go over pathologies in the ECS and CNS that cannabis, in study, has been proven to manipulate including the directives of cannabinoids, endogenous cannabinoids, and TRPV channels [1]. The ECS is a far-reaching neuromodulatory system having strong presence and significant roles in the CNS. The ECS consists of cannabinoid receptors, lipid-based retrograde neurotransmitters (endocannabinoids) heavily existing in the CNS including specific enzymes responsible for the synthesis and degradation of endocannabinoids.

#### **4.2 2-AG and anandamide**

2-Arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide) are the best-studied endogenous cannabinoids and are synthesized and degraded by distinct pathways. 2-AG is an agonist for either CB1 or CB2 receptors. Interestingly, anandamide is a low-strength agonist at CB1 receptors and very low agonist at CB2 receptors [30–32]. "Implying systems with low receptor expression or when receptors couple weakly to signaling pathways anandamide can antagonize the effects of more efficacious agonists in efforts to maintain a directed homeostasis [33]." CB1 and CB2 receptors are primarily mediated by endocannabinoids, along with Transient Receptor Potential (TRP) channels. Primarily Anandamide degradation in the CNS is by the enzyme fatty acid amino hydrolase (FAAH) [34]. As its name suggests, FAAH degrades multiple fatty acid amides, including palmitoyl and ethanolamide. This has important experimental and therapeutic implications as inhibition of FAAH increases levels of these ethanolamides, which have widespread actions independent of cannabinoid receptors. It is important to note 2-AG and CB1 have interactions with serotonin

#### *Marijuana, a Journey through the Endocannabinoid System: Unmasking the Paradoxical Effect… DOI: http://dx.doi.org/10.5772/intechopen.101555*

via 5HT2C with a crucial participation from neuropeptide Y1 receptor (NPY1R) as explained in the article "Effect of cannabinoid-serotonin interactions in the regulation of neuropeptide Y1 receptors expression in rats: the role of CB1 and 5-HT2C receptor." Common precursors to the neurotransmitter serotonin, the hormone melatonin, and vitamin B3 are TRP channels specifically, TRPV1, that are activated by anandamide under certain conditions [35]. Anandamide also activates PPAR-alpha(responsible for cell division, cell growth, and cell death throughout life), a major overseer of lipid metabolism in the liver [32, 33]. PPAR-alpha goes active under energy deprivation and is necessary for the breakdown of fatty acids, which is a major adaptive response to prolonged fasting [36]. Moreover, increasing anandamide by decreasing its degradation by inhibition of FAAH also increases levels of other N-acylamides, in turn modulating PPARα [37, 38]. To explain, anandamide has practical roles in modulating and regulating pain, depression, appetite, memory, and fertility.

Importantly 2-AG biology, as an endogenous ligand for cannabinoid receptors like CB1 and CB2 in the brain, liver, and lung, and a major source of arachidonic acid, is used for prostaglandin synthesis [39]. Since 2-AG is an intermediate metabolizer in fatty acid synthesis [39], any manipulation of 2-AG production and degradation will undoubtedly have vast reaching effects that can even be independent of the ECS but interestingly avoiding the gut, heart, kidney, and spleen. A sound representative case is that "the measurement of bulk tissue levels of 2-AG is an indirect measure of 'synaptically-active' or 'interstitial' 2-AG, which is most relevant for cannabinoid receptor signaling and might be more accurately measured by microdialysis [40]."

Furthermore, sourced on knockout mice data, DAGLα, a prime enzyme responsible for 2-AG synthesis in the postsynaptic neuron in response to increased synaptic activity [41–43], appears to be the isoform responsible for most 2-AG production that contributes to synaptic plasticity in the adult CNS [32]. Many studies show 2-AG, Anandamide, CB1, CB2, and TRP channels naturally affecting serotonin either directly or indirectly. Whether they have a part in the paradoxical effect has yet to be analyzed.

#### **4.3 CB1 and CB2**

CB receptors have existed long before cannabis evolved circa 25 million years ago, beginning in organisms such as sea squirts and fugu fish 600 million years ago [44–46], but have evaded non-chordate invertebrates, i.e., insects, hydra, nematodes, fungi, and plants. CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) and their activation obstructs the catalyzing chemical reaction cyclases, voltagedependent calcium channels, activates several amino acids specific to the amino acids serine and threonine kinases, inwardly rectifying potassium channels, with some variation depending on cell type [45]. Thus, activation of CB1 or CB2 receptors exerts diverse consequences on cellular physiology, including synaptic function, gene transcription, cell motility, etc. [41]. CB1 receptors are exceptionally abundant in the cortex, basal ganglia, hippocampus, and cerebellum [47].

The majority of CB1 receptors are on nerve fibers, specifically axon terminals and pre-terminal axon segments, while avoiding the operational zones. Cortical and hippocampal CB1 receptor expression is particularly high on the direct pathway axons as they enter the globus pallidus heading toward the substantia nigra [48]. CB1 receptors are also expressed in glutamatergic neurons [49].

CB2 receptors in comparison with CB1 are expressed at much lower levels in the CNS. The CB2 receptor is primarily present in active immune defenses and vascular elements [50–52]. Interestingly, CB2 does appear to be expressed by nerve injury and has the potential to increase expression 100-fold post tissue injury or during inflammation [53]. It remains to be determined whether CB2 expression is increased in the CNS during brain injury. This is due to increased expression of CB2 on cells intrinsic to the CNS or is a result of the migration (e.g., CB2-expressing monocytes) of peripheral immune cells into the CNS.

Given that the paradoxical effect is found in the human and not the medicine, i.e., cannabis, it is important to shine light on the areas in which the ASR or ATD has functions. There are many studies that display the Endocannabinoid system manipulating serotonin/5HT. As understood in "Modulation of the Serotonin System by Endocannabinoid Signaling," serotonin can be actuated by the engagement of stress to constrain further activation of the HPA axis. The HPA axis is a group of closely knit influences and feedback interactions consisting of the Hypothalamic (CRH), Anterior Pituitary (ACTH), and Adrenal Cortex (CORT) and controls reactions to stress and regulates many body processes, including digestion, the immune system, mood and emotions, sexuality, and energy storage and expenditure. In turn the HPA axis is also under the control of the serotonergic system. Studies have shown that 5-HT through the activation of 5-HT receptors located in the PVN regulates neuroendocrine responses to stress (for review see, [54]). For instance, activation of the 5-HT1A receptors has been shown to reduce the secretion of ACTH (often produced in response to biological stress) and corticosterone (affecting carbohydrate, potassium, and sodium metabolism, i.e., glucocorticoid) induced by an array of stressors. The general consensus is that the serotonergic system contributes in the ECS-induced modulation of the HPA axis and stress responses. Researched receptors of the CNS and ECS that take part in Serotonin Modulation relevant to cannabis psychopharmacological effects are 5-HT1A, reducing stress (passive coping), and 5-HT2A, attuning actively or pro-actively through Default-mode-network/stress (active coping) as illuminated by RL Carhart-Harris & DJ Nutt in "Serotonin and brain function: a tale of two receptors" [14, 17, 55].
