The first identified endogenous component of the ECS was the first cannabinoid receptor (CB1) which was discovered and cloned from the cerebral cortex of rats (Matsuda et al., 1990). In the brain, CB1 receptors are the most common G-protein coupled receptor (GPCR), expressed mainly in neurons and varying greatly across brain areas (Mackie, 2005). Nonetheless, although in much lower concentrations, CB1 receptors are also present in astrocytes, oligodendrocytes and microglia (Stella, 2009; Castillo et al., 2012).

Similarly to most GPCRs, CB1 is essentially located in the cell membrane, particularly in presynaptic axon terminals. There, they are activated by ligands released by post-synaptic neurons upon their depolarization resulting in the inhibition of further neurotransmitter release (Szabo et al., 2000). This retrograde function can resolve into short-term depolarization-induced inhibition of excitatory or inhibitory (DSE and DSI, respectively) transmission (Yoshida et al., 2002; Diana and Marty, 2004), or long-lasting forms of neuroplasticity, like long-term depression (LTD) or potentiation (LTP) (Kellogget al., 2009; Silva-Cruz et al., 2017). Furthermore, CB1 can also act pre-synaptically leading to the activation of the mitogen-activated protein kinase (MAPK) pathway (Bouaboula et al., 1995), suggesting its involvement in cell proliferation and death processes in the hippocampus (Derkinderen et al., 2001). Lastly, CB1 has also been found in alternative subcellular localizations with different functionalities from their plasma membrane equivalents, constituting a subpopulation with distinct pharmacological properties (Robledo-Menendez et al., 2021).

Although the molecular mechanisms of CB1 require further investigation, several studies have linked CB1 function to certain behavioral pathways. Furthermore, CB1 knock-out (KO) mice display an anxiogenic phenotype (Ledent et al., 1999; Zimmer et al., 1999; Martin et al., 2002), suggesting a role for the ECS in anxiety. Interestingly, effects of CB1 agonists are broadly described as biphasic, since their activation in different types of cells can lead to opposing effects on behavior (Busquets-Garcia et al., 2018).

The cannabinoid receptor (CB2) was identified and cloned in 1993 (Munro et al., 1993). It was believed that CB2 was only expressed in immune cells, but their presence in the CNS was demonstrated years later (Van Sickle et al., 2005; Ashton et al., 2006). In light of such findings, an immunoregulatory function of CB2 was proposed. Transgenic models of mice lacking CB2 receptors have contributed greatly to the investigation of its immunomodulatory role. Indeed, CB2 KO mice show exacerbated inflammation (Turcotte et al., 2016). Such findings suggest the fundamental role of CB2 receptors in maintaining immune homeostasis across the organism.

In the CNS, CB2 receptors are present in microglia, macrophages, T and B cells, and natural killers (Matias et al., 2002; Klegeris et al., 2003; Graham et al., 2010; Ramirez et al., 2013). They are a key modulator of neurological activities like nociception, neuroinflammation, and neuroprotection (Malan et al., 2001; Cabral et al., 2008), while, at the same time, its activation is devoid of psychotropic effects. Although the cellular mechanisms of CB2 function are mostly unknown. CB2 are involved in neurological functions such as anxiety, impulsive behaviors, and pain (García-Gutiérrez et al., 2012; Navarrete et al., 2012; Han et al., 2013). Nevertheless, more research is required to clarify the function of CB2 in the CNS.

Even though during the past years numerous studies have investigated the ECS, to date, no further cannabinoid receptors have been reported. Although, some results suggest that certain effects of cannabinoids are not regulated by CB1 or CB2 (Brown, 2007), which has generated some debate regarding the potential existence of a third cannabinoid receptor, nonetheless this remains merely a hypothesis.

Following the identification of the endogenous cannabinoid receptors CB1 and CB2, research focused on the study of endogenous ligands. This lead to the discovery of the first cannabinoid-like substance N-arachidonoylethanolamide, also known as anandamide (AEA) (Devane et al., 1992). Additionally, a second endocannabinomimetic compound was isolated: 2-arachidonoylglycerol (2-AG). The discovery of these two endogenous cannabinoids (namely, endocannabinoids) reaffirmed the significance of the cannabinoid receptors and their ligands as mediators of a wide variety of biological mechanisms.

Both molecules are lipophilic structures derived from arachidonic acid (Pacher et al., 2006) and they are mainly generated at postsynaptic neurons. Their synthesis is triggered by an increase in postsynaptic intracellular calcium by itself or combined with the activation of postsynaptic GPCRs (Maejima et al., 2001). Upon release, endocannabinoids bind to CB1 and CB2 receptors in the presynaptic membrane with varying affinity. Specifically, AEA seems to be a high affinity, partial agonist of the CB1 receptor, but almost inactive at CB2, whereas 2-AG behaves as a full agonist at both CB1 and CB2 with moderate to low affinity (Sugiura et al., 2000; Pertwee et al., 2010; Di Marzo and De Petrocellis, 2012).

In the brain, the basal levels of 2-AG are approximately 200-fold higher than those of AEA (Sugiura et al., 2006), which indicates its more prominent effects in the CNS. 2-AG is mostly responsible for retrograde signaling via activation of CB1 receptors (Sugiura et al., 1997). It is considered the major mediator of CB1-induced forms of synaptic plasticity such as DSI and long-term hippocampal GABAergic depression (Wilson and Nicoll, 2001; Kim and Alger, 2004). Furthermore, 2-AG is able to activate CB1 receptors present in astrocytes, which eventually results in glutamate release (Navarrete and Araque, 2008) and therefore, mediating neuron-astrocyte communication.

Regarding AEA, it acts as a retrograde messenger and activates CB1 receptors expressed pre-synaptically in glutamatergic terminals (Grueter et al., 2010). This process results in LTD via suppression of glutamate release. Furthermore, AEA participates in “tonic” suppression of GABAergic transmission in the hippocampus (Kim and Alger, 2010). Besides, AEA can act on intracellular CB1 associated with endosomal and lysosomal compartments.

Overall, it is hypothesized that, regarding CB1, AEA represents the “tonic” signaling molecule regulating basal synaptic transmission, whereas 2-AG represents the “phasic” signal which is activated during sustained neuronal depolarization and is responsible for many forms of synaptic plasticity, at least, in the hippocampus (Castillo et al., 2012; Lee et al., 2015). However, these signaling pathways might differ in other brain regions under different physiological or pathophysiological conditions.

When bound to CB2 receptors, endocannabinoids display a series of effects in immune cell function. Interestingly, there is a pronounced contrast between the effects of AEA and 2-AG on immune regulation. 2-AG was found to regulate mechanisms regarding leukocyte recruitment like chemokine release and cell migration (Turcotte et al., 2016). This implies the existence of positive regulation of the immune system by 2-AG. Alternatively, AEA was shown to downregulate leukocyte functions, such as cytokine release and nitric oxide production (Turcotte et al., 2016). Some studies report an increase in production of anti-inflammatory molecules like interleukin (IL) 10 in cells treated with AEA (Correa et al., 2010; 2011). In fact, CB2 receptor agonists like delta-9-tetrahydrocannabinol (THC) and WIN 55,212-2 have only been shown to cause anti-inflammatory effects on leukocytes (Turcotte et al., 2016).

AEA and 2-AG have also been reported to interact with other receptors in the body. AEA acts as an endogenous ligand for transient receptor potential vanilloid 1 (TRPV1) (Starowicz et al., 2007). At pre-synaptic TRPV1s, AEA directly facilitates glutamate release in the striatum (Musella et al., 2009). Alternatively, post-synaptic activation of TRPV1 by AEA also occurs and results in a reduction of biosynthesis of 2-AG (Maccarrone et al., 2008) which, in turn, might lead to LTD (Chávez et al., 2010). In addition, AEA receptor targets include other GPCRs like GPR55 and GPR119 (Sharir et al., 2012). However, these interactions have not yet been fully studied nor understood.

Other key components of the ECS are the catabolic and anabolic enzymes responsible for the synthesis and degradation of endocannabinoids. Briefly, 2-AG is produced by the enzyme diacylglycerol lipase (DAGL) starting from the compound diacylglycerol (DAG) (Murataevaet al., 2014). Alternatively, the biosynthesis of AEA begins from membrane phospholipid precursor N-acyl-phosphatidylethanolamine (NAPE) and it involves the enzymes calcium-dependent or independent N-acyltransferase (NAT or iNAT, respectively) (Jin et al., 2007; Jin et al., 2009) together with NAPE-specific phospholipase D (NAPE-PLD) (Okamoto et al., 2004). However, the exact mechanisms of its synthesis remain under investigation.

Regarding their catabolism, both molecules follow distinct paths. Upon 2-AG reuptake, it is degraded by enzyme monoacylglycerol lipase (MAGL) (Dinh et al., 2002, 2004) which can be found in presynaptic locations and in axon terminals (Gulyas et al., 2004). On the other hand, AEA is degraded by fatty acid amide hydrolase (FAAH) into arachidonic acid and ethanolamine (McKinney and Cravatt 2005) present, particularly, in postsynaptic terminals (Gulyas et al., 2004).

Importantly, suppression of FAAH and MAGL leads to an activity prolongation of endocannabinoids (Gaetani et al., 2009) resulting in differential effects. On one hand, blocking anandamide degradation reduces pain, inflammation depression and anxiety (Tanaka et al., 2019; Y.; Wang and Zhang, 2017). On the other hand, blockage of 2-AG degradation leads to hypothermia, hypomotility and analgesia (Long et al., 2009). This observation suggested FAAH as a potential pharmacological target and the development of synthetic inhibitors that could potentiate AEA transmission which might prove beneficial for the treatment of disorders like MDD (Fowler, 2015). A schematic representation summarizing the role of the named elements of the ECS in neurotransmission can be observed in Figure 1.

Phytocannabinoids are cannabinoids produced in the trichomes of the Cannabis plant (Cannabis sativa, Cannabis indica and Cannabis ruderalis). These species contain more than 100 different biologically psychoactive compounds (Aizpurua-Olaizola et al., 2016; Pertwee, 2006). Among them, the most commonly studied due to its strong psychotropic effects is THC. Nonetheless, an increasing number of studies have focused on a different compound named cannabidiol (CBD) since it displays very different pharmacological effects than THC without psychotropic activity. Importantly, later findings unveiled the lack of psychotropic activity of CBD altogether, as well as its absence of reinforcing and addictive properties and a number of characteristics which will advocate for its high potential for therapeutic use (Howlett et al., 2002). Interestingly, the pharmacological effects of CBD go beyond endocannabinoid receptors. For instance, when exerting activity at 5-HTA receptors, CBD induces anxiolytic effects (de Mello Schier et al., 2014; Zanelati et al., 2010). Furthermore, CBD can act as an agonist of vanilloid receptor TRPV1 and therefore influences pain perception, inflammation (Costa et al., 2004). Beyond that, CBD is also known to have affinity for glycine (Ahrens et al., 2009), GABA_A (Bakas et al., 2017), adenosine A1 (Gonca and Darıcı, 2015) and nuclear receptors (Scuderi et al., 2014).

In this manner, research then established that THC mainly acts as a partial agonist of CB1 and CB2 (Shahbazi et al., 2020). Alternatively, CBD was found to have reduced binding affinity to both cannabinoid receptors (Pertwee, 2008). However, this compound can also act as a negative allosteric modulator of CB1 and CB2 receptor agonists (Laprairie et al., 2015).

Genetic studies are a great source of evidence confirming the role of the ECS in MDD. Together with other genetic factors external to the ECS, epidemiological work of past decades has provided evidence of a certain degree of MDD heritability (Mondimore et al., 2006). Hence, it was hypothesized that variations in certain genes could be substantially responsible for the development of MDD. Nonetheless, up to date, no single genetic mutation is necessary nor sufficient to explain MDD, instead, each gene variation contributes only a reduced fraction of the total risk (Major Depressive Disorder Working Group of the Psychiatric GWAS Consortium et al., 2013).

In humans, genetic studies have investigated single nucleotide polymorphisms in the CB1 gene (CNR1) associated to depressive phenotypes and responses to antidepressants. For example, presence of the G allele of rs806371 CNR1 gene polymorphism is higher in individuals with MDD (Mitjans et al., 2013). On the same line, the G allele of the CNR1rs1049353 polymorphism has been associated to antidepressant resistance (Domschke et al., 2008). Alternatively, the minor C allele of rs2023239 displayed a protective influence against MDD (Icick et al., 2015).

Nevertheless, studies associating gene variants and MDD often encounter inconsistent data. A recent report analyzed several candidate genes previously implicated in high prevalence of MDD, among them, a CNR1 variant (Gonda et al., 2018). It was observed that most polymorphisms, including those in CNR1, showed increasing relevance for MDD in participants with higher exposure to recent negative life events. Possibly, the lack of accountability for environmental factors like stress could bias their contribution to MDD and cause the results to become irreproducible. As a matter of fact, a recent meta-analysis by Kong et al. (2019) revealed no association between CNR1rs1049353 or CNR1 triple repeat with increased risk of MDD when combining all available literature.

In contrast, a significant correlation was found between all four genetic models of the CB2 gene (CNR2) polymorphism CNR2rs2501432 and MDD. This was first observed by Onaivi and collaborators (2008), who reported a significant association between such CNR2 polymorphism and depressed patients. Furthermore, a recent article has linked CNR2 R63Q variation to a greater sensitivity towards childhood trauma and overactivation of the HPA-axis (Lazary et al., 2019). Interestingly, the article also describes the involvement of a FAAH gene polymorphism in the susceptibility to trauma observed in Lazary et al. (2016).

In conclusion, the role of CNR1 in MDD should not be dismissed and further research with wider coverage is needed to evaluate its impact. However, this finding positions CNR2 as a key factor in the development of MDD. Hence, pharmacological modulation of CNR2 ligands might be a potential therapeutic approach for this disorder. Importantly, the association of endocannabinoid receptors to MDD is not sufficient to explain the entirety of its pathophysiology. Therefore, such results should be interpreted with caution, while contemplating the essential role of environmental factors in its etiology.

Stressful life events are considered the main predisposing factor for the development of psychiatry disorders like anxiety and MDD. In mammals, the stress response is mediated by the HPA-axis, resulting in a cascade of events involving a series of hormones; like corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH); ultimately resulting in the release of glucocorticoids into the bloodstream (Smith and Vale, 2006). Termination of the stress response is achieved by resetting the HPA-axis via negative feedback mechanisms. On this basis, hyperactivity of the HPA-axis has been suggested as a mechanism leading to stress vulnerability shown by MDD patients. Certainly, these patients show inadequate HPA-axis suppression when exposed to stress or exogenous glucocorticoid administration compared to healthy controls (Gillespie and Nemeroff, 2005; Juruena, 2014). Interestingly, this dysregulation of the HPA-axis seems to be related to endocannabinoid signaling (Hillard et al., 2016).

For example, CB1 receptors are believed to play a major role in HPA-axis regulation. This hypothesis arose from the observation of high basal corticosterone levels and HPA hyperactivity in CB1 KO animal models (Barna et al., 2004). This indicated a clear role for CB1 in the inhibition of the HPA-axis and therefore, in the termination of the stress response. On the other hand, HPA-axis dysregulation can also influence the ECS via alterations in endocannabinoid synthesis and CB1 expression, both involved in stress-related symptoms (Micale and Drago, 2018). This suggests the existence of a bidirectional link between the two systems.

Furthermore, the role of the ECS was investigated in existing stress models associated to anxiety-like behavior in rodents. It was observed that chronic stress induced a reduction of AEA levels in the amygdala and hippocampus (Patel et al., 2005; Wang et al., 2012) via increased activity of FAAH (Navarria et al., 2014; Gray et al., 2015), which is consistent with previous evidence supporting the anxiolytic properties of AEA (Patel and Hillard, 2006). This reduction was attributed to the elevated serum corticosterone content (Hill et al., 2009) through a CRHR1-mediated mechanism (Gray et al., 2016) resulting in the generation of anxiety (Gray et al., 2015). Nevertheless, the obtained results depended largely on the type of stressor. Furthermore, exposure to repeated stressors increased 2-AG content throughout regions in the CNS (Micale and Drago 2018) probably mediated by decreased expression of MAGL (Sumislawski et al., 2011). This is in accordance with the observed decrease in AEA levels following stress exposure, since its interaction with TRPV1 results in downregulation of 2-AG synthesis (Maccarrone et al., 2008). However, this link has not been empirically demonstrated.

Importantly, following chronic unpredictable stress, CB1 undergoes widespread downregulation and desensitization in limbic areas like the hippocampus, hypothalamus, amygdala and nucleus accumbens (Wamsteeker et al., 2010; Wang et al., 2010; Lee and Hill, 2013). In contrast, many studies have found an upregulation of CB1 in PFC (Zoppi et al., 2011; Lee and Hill, 2013). Unfortunately, the exact mechanism by which this CB1 regulation occurs remains under speculation. However, it is consistent with the observation that CB1 activation decreases GABAergic transmission in said limbic areas, contributing to the termination of the stress response (Martin et al., 2002; Hill et al., 2011). Particularly, CB1 blockade leads to increased plasma corticosterone (Wade et al., 2006). Furthermore, the activation of CB1 on noradrenergic signaling represents an important mechanism for stress adaptation (Wyrofsky et al., 2019). Chronic HPA-axis-induced stimulation of noradrenergic areas such as the locus coeruleus increases anxiety and depressive-like behavior (Valentino and Van Bockstaele, 2008). Importantly, hyperactivity of the ECS via CB1 signaling due to chronic stress might lead to increased noradrenergic activity in the PFC and thus, contributing to such behaviors (Wyrofsky et al., 2019).

On this basis, CB1 dysregulation within specific brain areas would lead to a hyperactive HPA-axis, which is precisely what is observed in CB1 KO animals (Daniela Cota et al., 2007; Steiner et al., 2008). CB1 KO animals express higher anxiogenic-like behavior and increased stress-induced ACTH levels, as well as anxiolytic drug resistance (Cota, 2008). Accordingly, hyperactivity of the HPA-axis is one of the most consistent biological evidence in MDD in both clinical and pre-clinical studies (Sánchez et al., 2001; Heim and Nemeroff 2002). This suggests a clear role for CB1 as a direct contributor to the termination of the HPA-mediated stress response and therefore, as a key partaker in the pathophysiology of depressive symptoms.

With regards to CB2 receptors, despite their expression in areas involved in the stress response, few studies have explored their implications in HPA-axis signaling. In these, results are contradictory. Some stress-induced depressive-like behavior models have described no changes in CB2 levels, whereas others encountered a decreased CB2 hippocampal expression (Marco et al., 2017). However, unlike CB1, manipulation of CB2 receptors does not alter plasma concentrations of corticosterone after exposure to stress (Zoppi et al., 2014). Such findings speak against a direct relationship between the HPA-axis and CB2 receptors, although it is possible that CB2 might regulate the activity of the HPA-axis through other indirect mechanisms. Indeed, a recent article has linked an existing polymorphism of the gene encoding CB2 to greater sensitivity for childhood trauma and overactivation of the HPA-axis (Lazary et al., 2019). This process seems to be mediated through neuroinflammatory mechanisms influenced by CB2. This observation is in accordance with previous findings showing that CB2 KO animals display exacerbated stress-influenced neuroinflammatory responses (Zoppi et al., 2014).

All together, these findings suggest the involvement of the ECS; particularly, the CB1 receptor and endocannabinoids AEA and 2-AG, as a fundamental regulatory system involved in the termination of the stress response by mediating negative feedbacks controlling HPA-axis’ activity. Therefore, these ECS components have a direct role in anxiety-like behavior and vulnerability to stress, and they must be regarded as important contributors to the pathophysiology of MDD. All the above-mentioned mechanisms are graphically described within the blue area of Figure 2.

Among the other potential altered circuitries in MDD, neuroinflammation has received increasing attention (Troubat et al., 2020). Since the first observations, several studies have described a strong relationship between depressive symptomology and altered presence of pro-inflammatory markers. Hence, research has focused on the involvement of the immune system in the CNS, which consists of glial cells.

As the resident macrophagic cells and main form of innate immune defense in the CNS, microglia were expected to play a crucial role in this relationship. Microglia are responsible for the release of chemokines and cytokines as a response to physical insults or infectious agents. Upon activation, microglia can evolve into two possible phenotypes, known as M1 and M2, which might include specific features under different pathological insults (Ma et al., 2017). The first, M1, is in charge of the initiation of the inflammatory response through the release of pro-inflammatory mediators like IL-1β, IL-6, IL-8 and tumor necrosis factor α (TNFα) (Lively and Schlichter, 2018). Furthermore, they control the recruitment of additional central and peripheral immune cells to the site of infection or brain damage. Alternatively, the second phenotype, M2, is involved in the prevention of M1-induced neuronal damage and toxicity through secretion of anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor (TGF) (Koscsó et al., 2013). This M1/M2 phenotype theory has been found to be an oversimplification of this cytological process based on experimental evidence (Ransohoff 2016). However, this terminology is useful when characterizing microglia states (pro-inflammatory and neuroprotective) and will continue to be used for the purpose of this review.

Due to their primary role in neuromodulation of the inflammatory response, microglia have become the center of attention as a key component in the inflammatory etiology of MDD (Yirmiya et al., 2015). Evidence supporting these theories is provided by clinical and preclinical research. For instance, elevated levels of proinflammatory cytokines released by microglia are correlated to the severity of depressive symptomatology in MDD patients (Haapakoski et al., 2015). Furthermore, rodents exposed to chronic stress paradigms display microglia hyper-ramification and overactivation in several brain regions (Sugama et al., 2007; Tynan et al., 2010; Hinwood et al., 2013).

Particularly, IL-6 and C-reactive protein (CRP) are the most strongly associated to anhedonia (Felger et al., 2018). In accordance, administration of antidepressants directly impacts microglia signaling and IL-6 production (Hashioka et al., 2007). A more recent study has also linked the imbalance of microglial pro- and anti-inflammatory conditions through a mechanism involving brain-derived growth factor (BDNF), more specifically, via a BDNF-TrkB-dependent pathway in the hippocampus (Liu et al., 2019). Interestingly, the authors found a sex-specific relationship between BDNF and microglial inflammatory biomarkers, a mechanism that could become very important for sex differences in depression. On this basis, some researchers have suggested that MDD could start to be considered a microglial disease (Yirmiya et al., 2015).

The presence of CB2 receptors in microglia switched the attention to the potential role of the ECS in glia-mediated neuroinflammation involved in MDD. CB2 has been described to possess general anti-inflammatory properties. Genetically modified rodents with overexpression of CB2 show reduced stress-induced pro-inflammatory cytokine TNFα and enzyme COX-2 (Zoppi et al., 2014). In parallel, CB2 KO animals display exacerbated neuroinflammatory responses to chronic stress (Zoppi et al., 2014).

CB2 receptors are expressed in microglia in a state-dependent manner (Carlisle et al., 2002). Specifically, they are not present in resting microglia but rather in fully active cells and intermediary stages. In the past decade, its presence has been associated to induction of M2 polarization (Tanaka et al., 2020). For example, administration of synthetic CB2 agonists alleviate neuroinflammation by enhancing the conversion from M1 microglia into the M2 phenotype (Tao et al., 2016; Luo et al., 2018). Importantly, a downregulation of inflammatory cytokines and upregulation of anti-inflammatory mediators were paired to M2 polarization (Tao et al., 2016). These mechanisms were not observed when animals were co-treated with CB2 synthetic antagonists. Such results evidence the seemingly important role of CB2 receptors in microglia-induced neuroprotection in the presence of inflammation.

Furthermore, it appears these anti-inflammatory effects of CB2 might be initiated by AEA. Administration of AEA reduces neuron toxicity by downregulating IL-1B and IL-6 in in vitro activated microglia via CB2 receptors (Malek et al., 2015). At the same time, it exerts neuroprotective properties via production of IL-10 (Correa et al., 2010), and upregulation of CD200 receptor, known to suppress microglial inflammatory response (Manich et al., 2019). Moreover, pharmacological inhibition of FAAH also leads to anti-inflammatory effects in vitro via increased levels of AEA (Tanaka et al., 2019).

Although these observations have been widely described in pharmacologically induced neuroinflammation, the immunological role of the ECS in the context of MDD models is understudied. However, two recent studies have investigated this relationship. First, Chen et al. (2018) reported that FAAH inhibition resulted in alleviation of pro-inflammatory response to acute stress. This is in accordance with previous studies reporting anti-inflammatory properties of AEA. Second, Lisboa et al. (2018) observed that administration of selective cannabinoid agonist WIN55,212-2 was able to reverse social stress-induced neuroinflammation and anxiety-like symptomatology. Although, since named agonist could act on both cannabinoid receptors, whether this action occurred through mediation of CB1 or CB2 is unclear.

Regarding CB1, despite its low expression in glial cells, some studies have shown that this receptor is also able to regulate immune function through distinct mechanisms. For instance, specific CB1 KO of forebrain GABAergic neurons in vivo lead to pro-inflammatory microglia phenotypes without significant cognitive deficits (Ativie et al., 2018). This finding provides evidence of the role of CB1 in neuron-glia communication with a crucial involvement of GABAergic neurons. Furthermore, a recent research work observed that chronic stress increased microglia activation in CB1 KO animals, which correlated with the severity of depressive-like symptoms (Beins, 2020). Hence, it has been hypothesized that CB1 might be an indirect mediator of microglia-induced inflammation. Although, how CB1 signaling can regulate microglial activity remains uncertain and requires further research.

For this, it is important to consider the potential interplay between HPA-axis and microglial neuroinflammation. As mentioned earlier, HPA-axis hyperactivity is observed across animal models of MDD, as well as in human patients. Increased glucocorticoid release has been found to induce an overproduction of pro-inflammatory cytokines through microglia activation (Nair and Bonneau, 2006; Sorrells and Sapolsky, 2007). In the same manner, an immune modulation of the HPA-axis has also been suggested following immunology research of viral infections (Silverman et al., 2005). However, the exact mechanisms underlying chronic stress and microglia activation remain under discussion. A recent article described a potential link between HPA signaling and neuroinflammation. Feng et al. (2019) observed that elevated corticosterone levels after chronic stress increased the release of pro-inflammatory elements like IL-1β and IL-18 in activated hippocampal microglia. Particularly, they observed that this process was mediated by nuclear factor kappa B (NF-κB) and the Nod-like receptor protein (NLRP3) namely, NF-κB-NLRP3 pathway. This study provides a new and valuable insight into the relationship between stress, the HPA-axis and microglia-mediated neuroinflammation.

Although much less investigated, the ECS might also be related to this mechanism. Recent research has found that administration of two phytocannabinoids; CBD and cannabigerol produced anti-inflammatory effects by reducing NF-κB activation among other mechanisms (Mammana et al., 2019). Furthermore, previous work had stablished a neuroprotective role for CB2 via inhibition of the NLRP3 inflammasome in autoimmune encephalomyelitis models (Shao et al., 2014). This process might be mediated via the CB2-induced inhibition of NF-κB activation in microglia (Zoppi et al., 2014). Additionally, CB1-mediated effects of 2-AG have been linked to neuroprotective functions via inhibition of NF-κB (Panikashvili et al., 2005). Likewise, AEA has been described to enhance production of IL-10 in activated microglia and inhibiting NF-κB activation. More recently, in the context of liver inflammatory disease, CB1 receptors have been reported to mediate macrophage NLRP3 expression and inflammation (Yang et al., 2020). This evidence suggests a potential role for the ECS as a bridge in the crosstalk between the HPA-axis and neuroinflammation via the NF-κB-NLRP3 pathway. However, effects of the ECS in modulation of microglia-mediated neuroinflammation as a result of chronic stress, particularly regarding the NLRP3, remains to be studied and requires much further research.

Importantly, a novel field of research has emerged in recent years, focusing on polyunsaturated fatty acids (PUFAs) and their potential as a treatment for MDD. PUFAs are lipid derivatives of omega-3 or omega-6 that act as precursors for endocannabinoids. Particularly, supplementation with two main PUFAs, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been extensively investigated (Mozaffari-Khosravi et al., 2013; Marangell et al., 2003). Indeed a recent meta-analysis confirmed the beneficial effects of omega-3 PUFAs on depression symptoms (Liao et al., 2019). Co-administration of DHA and EPA or EPA alone are able to reduce inflammation through several mechanisms. Firstly, they both can reduce the production of proinflammatory cytokines like TNFα, IL-1β, IL 2 and IL-6 (Caughey et al., 1996). Secondly, they have been described to suppress NF-κB signaling (Horowitz et al., 2015). Lastly, administration of a variety of omega-3 PUFAs reduced inflammation via activation of macrophage autophagy and attenuation of NLRP3 inflammasome (Shen et al., 2017). These findings strongly confirm the endocannabinoid regulation of neuroinflammatory processes, and they provide an opening to a new research field in the context of psychiatric disease like MDD.

Overall, the abovementioned evidence supports the theory that hypofunction of the ECS directly impacts neuro-immune modulatory pathways in the CNS, leading to pro-inflammatory processes mediated by microglia. Specifically, CB2 seems to be directly involved in promotion of anti-inflammatory mechanisms mediated by AEA signaling. Nevertheless, the involvement of CB2 in stress-induced inflammation remains unknown. Alternatively, CB1 has been suggested to indirectly modulate neuro-immune responses by mediating neuron-glia communication in the CNS. Such interactions between the immune system, the ECS and MDD symptomatology are depicted in the yellow area in Figure 2.

Adult neurogenesis is a neurobiological process by which neurons are continually generated within the CNS throughout an organism’s life. Specifically, the subgranular zone (SGZ) of the dentate gyrus of the hippocampus has been established as the primary area involved in this process (Bond et al., 2015). Notably, the exact biological role of neurogenesis in cognition and behavior remains unknown.

Since the discovery of hippocampal atrophy in untreated MDD patients (Sheline et al., 1996), it was hypothesized that loss of neurogenesis in the dentate gyrus could significantly impact the development of this disorder. In fact, successful treatment with antidepressants and most therapies is shown to induce hippocampal neurogenesis (Praag et al., 2000; Malberg and Duman 2003; van). This phenomenon seems to be mediated by BDNF, since it is found to be reduced in animals models of MDD (Autry et al., 2009), while simultaneously recovered by antidepressant administration (Santarelli et al., 2003). However, neurogenesis ablation in experimental animals does not always induce depressive symptoms (Jayatissa et al., 2010). On this line, some antidepressants show neurogenesis-independent mechanisms (David et al., 2009). Due to such conflictive results, it is currently hypothesized that neurogenesis might be a key restorative mechanism for hippocampal structure and function which might indirectly result in alleviation of MDD symptomatology (Hanson et al., 2011). Therefore, when disrupted, it could theoretically participate in the etiology of MDD even though it is unlikely to cause the entire mood disorder. In the present, however, neurogenesis is still considered a significant contributing factor to the pathophysiology of MDD (Jesulola et al., 2018).

During the past decades, research has confirmed the involvement of the ECS in hippocampal proliferation, starting with the extensive expression of endocannabinoid entities in neuronal progenitor cells (Aguado et al., 2006). Specifically, CB1 has been established as a direct mediator of adult neurogenesis. For example, administration of high selective agonists of CB1 promotes neural proliferation in the SGZ (Andres-Mach et al., 2015). Accordingly, this effect was prevented by CB1 synthetic antagonist AM251 and not present in CB1 and CB2 double KO animals (Hutch and Hegg 2016). Furthermore, DAGLα KO rodents exhibited decreased 2-AG levels, as well as impaired neurogenesis in the dentate gyrus (Gao et al., 2010). Therefore, CB1 activation via 2-AG agonism seems to play a major role in this biological process. However, the exact mechanism by which CB1 might induce neurogenesis is under discussion, although some pathways have been described (Zimmermann et al., 2018).

Relevant to depressive-like behavior, CB1 KO mice were observed to be more vulnerable to stress-induced depressive-like responses with a high susceptibility for anhedonia (Martin et al., 2002). This symptomatology was later associated to downregulation of BDNF expression in the hippocampus (Aso et al., 2008). Such findings suggested a clear involvement of both, CB1 and neurogenesis in the development of depressive disorders.

Conversely, administration of CBD prevented detrimental effects of chronic stress and increased hippocampal proliferation via CB1 activation (Wolf et al., 2010; Campos et al., 2013). Another recent study reported that CBD administration could reverse the effects of chronic stress, facilitating neurogenesis and dendritic remodeling (Fogaça et al., 2018). However, whether anxiolytic effects of CBD are directly a consequence of improved neurogenesis or other unrelated endocannabinoid mechanisms cannot be concluded. These observations also occurred when considering endocannabinoids. For example, prevention of 2-AG degradation via blockade of MAGL results in an enhancement of neurogenesis and heightened antidepressant-like effects in mice exposed to chronic stress (Zhang et al., 2015). In addition, DAGLα KO animals displayed reduced endocannabinoid levels in the hippocampus, as well as impaired neurogenesis and anxiety-like behavior (Jenniches et al., 2016). Nonetheless, the cause-consequence relationship between the observed depressive symptoms and loss of neurogenesis cannot be established from this study. In summary, CB1 is hypothesized to contribute to anti-depressive effects paired with neurogenic mechanisms in the hippocampus.

A more complex role of CB2 in adult neurogenesis has been suggested. Unlike CB1 KO, CB2 KO animals display a stable adult neurogenesis (Mensching et al., 2019). Although, these findings are contradictory to previously reported observations indeed showing altered neurogenesis in this strain (Palazuelos et al., 2006). This perhaps could be due to the age difference among rodents used for the studies. Regardless of this contradiction, CB2 agonism has been related to indirect potentiation of neurogenesis through its role as a homeostasis regulator, normalizing processes like apoptosis, oxidative stress, and neuroinflammation (Avraham et al., 2014; Shi et al., 2017). For instance, in an Alzheimer’s disease model, administration of CB2 agonist MDA7 induced neurogenesis together with improved hippocampal synaptic plasticity and regulated microglial activation (Wu et al., 2017). Interestingly, microglia are able to regulate this process via the release of cytokines and chemokines that may act as stimulants or suppressors of neurogenesis (Sato 2015). In this manner, pro-inflammatory mediators like IL-1β can inhibit processes such as cell proliferation and differentiation in the dentate gyrus (Wu et al., 2013). In particular, chronic stress in rodents produces alterations in microglia activation, which contributes to the loss of neurogenesis (Kreisel et al., 2014). Alternatively, microglia can also release BDNF, thereby supporting survival of novel neurons (Ferrini and De Koninck 2013). Whether this mechanism is directly regulated by CB2 remains unclear. Nevertheless, more recently it has been observed that activation of CB2 receptors by selective agonist JWH133 upregulated microglia expression of BDNF in the lateral ventricular tissue (Tang et al., 2017). Though this finding has shed some light on the topic, further research is still required before drawing solid conclusions.

Neurogenesis is also related to HPA function in pathological conditions. For example, animals with suppressed neurogenesis display a hyperactive HPA-axis associated to depressive-like behaviors (Schloesser et al., 2009). Given the important role of the hippocampus in termination of HPA-mediated stress response, it was hypothesized that disturbances in neurogenesis could negatively alter the negative feedback loop regulating HPA activity. Simultaneously, chronically elevated glucocorticoid concentrations as a result of a hyperactive HPA also leads neurogenesis disruptions (Anacker et al., 2013).

Interestingly, changes induced by stress or inflammation may affect neurogenesis via the NF-κB pathway (Anisman et al., 2008), which is, at the same time, the suggested mechanism bridging HPA activity and neuroinflammatory processes. Furthermore, pro-inflammatory cytokines can additionally stimulate the HPA-axis to release glucocorticoids, which, in turn, suppress neurogenesis (Liu et al., 2003). Hence, it can be concluded that there is a strong interconnection between the ECS and altered mechanisms observed in MDD.

Overall, these results indicate a role for the ECS in modulation of hippocampal cell proliferation and cell differentiation. On one hand, CB1 and 2-AG signaling seem to be essential components in the stimulation of neurogenesis. On the other, CB2 might participate as a key regulator under pathological conditions by exerting neuroprotective mechanisms towards neurons via immune system regulation. These mechanisms are depicted within the red area in Figure 2. In conclusion, the presented data suggest a clear involvement of the ECS in the neuroprotective effects of hippocampal neurogenesis during the development of MDD.