Part 1: The Endocannabinoid System (ECS)
[ My Perspective ]
The endocannabinoid system (ECS) is the body’s spiritual system.
Spiritual growth can be defined as obtaining a profound insight that alters one’s thoughts and behaviors in a significant and sustained way. The ECS is the only biological system that can accomplish this objective.
With its ability to inhibit presynaptic neurotransmitter release (induce changes in synaptic plasticity) of both excitatory and inhibitory neurotransmission and modulate a wide range of neurotransmitter systems including dopamine and serotonin, combined with its ubiquitous presence, the ECS can induce long-term and/or short-term changes in multiple biological processes simultaneously. Thereby, achieving the behavioral outcome of a transformed being through top-down control/realization.
(Mediate: directly regulates)
v.s.
(Modulate: indirectly regulates)
[ The Traditional Perspective ]
The ECS is a complex cell-signaling system that acts as a central control of most, if not all, physiological and neurological processes, and thereby maintaining homeostasis in the body.
[ The Objective Perspective ]
The ECS is the conceptual grouping of the endocannabinoid receptors (CBRs), the endocannabinoids (eCBs), and the endocannabinoids metabolism enzymes. The ECS is present in all vertebrates including dogs, cats, birds, fish, lizards, frogs, and snakes (so if the ECS is truly the spiritual system, they are all capable of experiencing spiritual enlightenments/insights, which my snake indeed does).
Key components of the ECS:Endocannabinoid Receptors (CBRs)Endocannabinoids (eCBs)Endocannabinoids Metabolism Enzymes
[1] The Endocannabinoid Receptors (CBRs)
The endocannabinoid receptors (CBRs) (aka the cannabinoid receptors) are the binding site for both endocannabinoids (AEA, 2-AG, etc. - more on this later) and phytocannabinoids (THC, CBD, etc.). It is through binding with the CBRs that cannabis triggers various cascading processes and exerts its physiological effects.
While there are other CBRs, CB1R and CB2R are the two most well-known and well-studied CBRs. CB1Rs are primarily expressed in the central nervous system (CNS) — THC exerts its psychoactive effects through binding with the CB1Rs. Meanwhile, CB2Rs are primarily found in the peripheral nervous system (PNS). CB1Rs are also found in the PNS, but to a lesser extent, and CB2Rs are found to be up-regulated in the CNS under pathological conditions.
(CNS: the brain and the spinal cord.)
v.s.
(PNS: nerves and ganglia that stem out of the CNS to facilitate communication between the CNS and the rest of the body/peripheral tissues.)
(Nerves: bundles of axons and dendrites of neurons.)
v.s.
(Ganglia: clusters of neuronal cell bodies.)
Both CB1R and CB2R are G protein-coupled receptors (GPCRs), or protein receptors that are integrated into the lipid bilayer of cell membranes [1]. CB1R was first discovered in the rat brain in 1988 [2] and first cloned in 1990 [3]. CB2R was first discovered in the human immune system (in the tonsils) and first cloned in 1993 [4]. In fact, it is the discovery of THC (1942) and CBD (1940) that led to the subsequent identification and cloning of CB1R and CB2R [1, 5].
(GPCRs: aka heptahelical receptors or seven-transmembrane receptors due to its distinctive structural feature: the protein spans seven times across the cell membrane. Upon ligand binding and receptor activation, the GPCR goes through conformational change and subsequently activates intracellular G proteins. The activated G proteins modulate intracellular enzymes to further trigger various downstream signaling cascades, and ultimately, lead to specific cellular responses/changes in gene expression.)
CB1R
CB1R is encoded by the gene CNR1. CB1R is the most expressed (i.e. the most abundant in number) GPCRs in the brain [6].
Within the brain, CB1R expression is found in all brain regions. Some areas have higher levels and more densely distributed CB1Rs while others have lower and more widespread CB1Rs [7].
On a synaptic level, CB1R is primarily expressed in presynaptic neuronal nerve terminals. CB1R activation leads to the inhibition of neurotransmitter release in the presynaptic neuron (neuron that is expressing the activated CB1R). It is thus believed that the main function of CB1R is to regulate synaptic transmission by regulating the excitability/inhibitory (E/I) balance in the CNS, as CB1Rs are expressed at both glutamatergic and GABAergic synapses [8, 9].
(Glutamate is the principal excitatory neurotransmitter in the CNS [10].)
v.s.
(GABA is the principal inhibitory neurotransmitter in the CNS [11].)
This reduction in synaptic transmission can be either short-term/transient (in milliseconds to seconds) or long-term (in minutes or hours or longer). When CB1R-mediated short-term synaptic plasticity happens at excitatory glutamatergic synapses, it inhibits the release of glutamate and lead to depolarization-induced suppression of excitation (DSE). Whereas when CB1R activation happens at inhibitory GABAergic synapses, depolarization-induced suppression of inhibition (DSI) occurs as GABA release is inhibited.
On the other hand, when CB1R activation leads to long-term reduction in synaptic transmission at excitatory glutamatergic synapses, it induces long-term depression (LTD). When it happens at inhibitory GABAergic synapses, it leads to long-term potentiation (LTP) of the neural circuit involved.
To summarize: activated by cannabinoids (endogenous or exogenous), CB1Rs are negative regulators of presynaptic neurotransmitter release, which includes both excitatory and inhibitory neurotransmitters, and thereby, CB1R regulates the E/I balance in the CNS [12, 11]. This change in E/I balance can be either short-term or long-term, resulting in DSI/DSE and/or LTD/LTP, respectively.
Aside from neurons and interneurons, CB1Rs are also found on glial cells (i.e. astrocytes, oligodendrocytes, and microglia) and certain cell types outside the CNS (e.g. immune cells) [8, 9]. In addition to its localization on cell membranes, CB1Rs are also found in various subcellular organelles including the lysosomes, endosomes, and mitochondria [12, 3].
Mitochondrial CB1R (mtCB1R) activation can decrease cellular and neuronal energy metabolism [13, 14], which in turn, can lead to impaired synaptic transmission [15] and a subjective feeling of brain fog and/or fatigue. On the other hand, mtCB1R activation also shows neuroprotective effects such as increasing cell viability, reducing oxidative stress, preventing apoptosis, and improving mitochondrial functions in damaged mouse hippocampal neurons [16].
Astroglial CB1R activation by either eCBs or exogenous cannabinoids can indirectly promote excitatory synaptic transmission. Specifically, astroglial CB1R activation increases intracellular Ca2+ concentration, leading to the subsequent release of gliotransmitters such as glutamate and D-serine. These gliotransmitters can stimulate synaptic transmission of pyramidal neurons, likely through the activation of NMDARs (N-methyl-D-aspartate receptors), which requires a co-agonist, such as D-serine, along with glutamate to activate and/or AMPARs (α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptors), which only requires glutamate to activate [17, 18, 19, 20].
Brain Region With High Levels of CB1R
[ The Cerebral Cortex ]
[ Introduction ]
The cerebral cortex (aka the neocortex) is the outermost layer of the brain. It is often divided into six layers (layer I to layer VI) and several anatomical, and to some extent, functional regions: the frontal lobe, parietal lobe, temporal lobe, occipital lobe, limbic lobe, insular cortex, and cingulate cortex. The primary sensory (somatosensory, visual, auditory, olfactory, and gustatory) and motor cortices and the association sensory and motor cortices also reside in the cerebral cortex.
[ CB1R Distribution ]
Using human and monkey brain samples, CB1R level is found to be higher in the neocortical association regions compared to the primary sensory cortices and the primary motor cortex [21]. Specifically, Brodmann area 46 (BA46) in the dorsolateral prefrontal cortex (dlPFC), which roughly occupies the middle third of the middle frontal gyrus, exhibits the highest CB1R density across all examined neocortical regions [21, 22]. However, there is also contradictory finding where CB1R level is found to be higher in the primary and association auditory cortices than in the prefrontal cortex (PFC) [12].
[ The Dorsolateral Prefrontal Cortex (dlPFC) ]: Neocortical Region With The Highest Level of CB1R
[ Introduction ]
The dlPFC is a specific region of the PFC. It is located in the middle frontal gyrus of the PFC, encompassing BA46 and BA9. Since the dlPFC is often functionally defined, its specific cutoff varies across individuals and studies. As a result, it can sometimes also include parts of BA8 and BA10 [23]. The dlPFC is one of the last brain regions to fully develop, but also one of the first to undergo age-related atrophy/brain size reduction [24].
[ CB1R Distribution ]
CB1R is found to be most densely distributed in BA46 in the dlPFC. Within BA46, the inner granular layer (layer 4) has the highest CB1R expression level. The inner granular layer contains mostly stellate neurons, and they primarily receive sensory inputs from the thalamus, the brain’s sensory information relay center. Cell type-wise, inhibitory PV+ interneurons that also express CB1R are found throughout layers 2-6 of BA46, with layer 4 showing the most prevalence [22].
(PV+ Interneurons: a specific type of inhibitory GABAergic interneurons characterized by the expression of parvalbumin (PV); PV+ interneurons release GABA and PV to form fast-spiking, high-frequency, and precise inhibitory synapses, and these synapses usually target the perisomatic regions (cell bodies and proximal dendrites) of excitatory neurons.)
(P.S. The “+” means the interneuron expresses PV, or shows positive testing for PV, depending on the specific (staining) method used.)
[ Functions ]
The dlPFC is critically involved in domain-general/non-task specific, goal-oriented executive functions, and its activity is often associated with some aspects of one’s general intelligence [23].
The dlPFC plays a superordinate role over its connected cortical and subcortical regions: it can either activate/further excite or inhibit relevant regions to achieve a specific objective, and this executive control function of the dlPFC seems to be lateralized [23]. The left dlPFC plays a more active role in attentional control (i.e. the active focusing of attention on task-/goal-relevant stimuli), while the right dlPFC serves as a broader, more passive attentional monitoring system over the external environment.
Excitatory/anodal high-definition transcranial direct current stimulation (HD-tDCS) over the left dlPFC during a (visual) attentional control task causally increased the theta-band activity of the right fronto-visual network compared to HD-tDCS over the right dlPFC, and consequently, led to increased attentional task performance (faster reaction time) in the presence of distractors [25].
Excitatory/anodal tDCS over the right dlPFC increased the detection of performance errors, whereas anodal tDCS over the left dlPFC showed no such effect [26]. Moreover, right dlPFC atrophy is associated with slower reaction time on accurate trials during a task of selective attention and inhibitory functions (the Flanker Task), suggesting a deficit in attentional monitoring [27].
(Anodal: the application of positive charge/current through the anode electrode; anodal tDCS depolarizes neurons, and thus, making them more excitable/more likely to fire an action potential [28, 29].)
v.s.
(Cathodal: the application of negative charge/current through the cathode electrode; cathodal tDCS hyperpolarizes neurons, and thus, making them less excitable/less likely to fire an action potential [28, 29].)
v.s.
(Sham: the control condition where a few seconds of stimulation that does not alter neuronal excitability is applied at the beginning and end of the experimental period in order to mimic the experimental conditions (anodal/cathodal, depending on the study [28, 29].)
The dlPFC is involved in, but not limited to, the following executive functions:
[ Working Memory (WM) ]: the mental maintenance and manipulation of information over a short period of time.
The dlPFC is consistently and persistently activated during the period requiring the use of WM for WM tasks (i.e. the retention period in delayed response tasks) [30].
Executive Control for WM Processes: the dlPFC acts as the executive central control to direct mental energy towards the most relevant WM content by enhancing the neural activities/mental representations of the most relevant WM content in its corresponding sensory region.
In the multi-component model of WM (the most prevalent WM model in contemporary cognitive neuroscience [31]), WM is broken down into three components: the left hemispheric verbal storage system (aka the “phonological loop”) for storing verbal information, where its capacity is limited by the number of verbal sequences that can be repeated before the first unit starts to fade away; the right hemispheric visual storage system (aka the “visuospatial sketchpad”) for maintaining non-verbal visual stimuli, where its capacity is also limited by one’s ability to rehearse and retrieve before the information starts to dissipate; and an executive central control component [32].
It is generally agreed that the dlPFC acts as this executive central control for WM [23], while the related sensory region stores high-resolution mental representations of the WM content. For example, the superior temporal gyrus, which includes the primary auditory cortex, is more activated during auditory than visual WM task. The posterior brain regions, which contain the primary visual cortex, and the anterior cingulate are more activated during visual than auditory WM task [33]. Whereas the lateral prefrontal cortex (lPFC), which includes the dlPFC, simultaneously considers multiple mental representations of goal-related factors to determine the relevance of different WM stimuli, and subsequently, the sets of neural activities to accentuate in the related sensory regions [34].
Distraction Elimination: the dlPFC is also responsible for enhancing the relevant working memory content when distractions are present, and thereby, acting as a top-down control for distraction elimination.
During a visual WM task, the dlPFC is directly stimulated with excitatory time-locked transcranial magnetic stimulation (TMS) during the retention period at the time point where a distractor could appear. TMS to the dlPFC increased the neural activities of the relevant WM content in its corresponding region, but not in that of the distractor, and dlPFC-TMS only further increased the neural activities representing the relevant WM content when a distractor appeared. dlPFC-TMS showed no significant effect on the relevant WM brain region when no distractor was present [35]. (Causal Evidence)
[ Emotional Regulation ]
Reappraisal: redefining the meaning of a negative emotional event (memory or experience) to optimize its emotional impact (e.g. interpreting a traumatic event as an opportunity for growth instead of a hindrance to one’s life). Reappraisal is central to mental well-being and is considered one of the most effective emotional regulation strategies. Deficiency in reappraisal is associated with psychiatric illnesses [36, 37].
Anodal transcranial direct current stimulation (tDCS) over the right dlPFC enhanced the reappraisal of personal negative emotional memories: participants in the active dlPFC-tDCS treatment group showed the greatest reduction in self-reported negative valence (valence: the intrinsic rating of attractiveness/aversiveness) of their negative emotional memories compared to other groups [36]. (Causal Evidence)
Cathodal continuous theta burst stimulation (cTBS) over the right dlPFC impaired reappraisal. Specifically, the reduction in the amplitude of Late Positive Potential (LPP) is decreased following the inhibitory stimulation sessions [37]. (Causal Evidence)
(LPP is a neural marker for stimulus-related emotional arousal: a reduction in its amplitude indicates reappraisal. Therefore, less amplitude reduction signifies less emotional reappraisal.)
Top-down Control of Affective States: the generation, and thereby, experiencing of emotional states via cognitive control.
The dlPFC is highly active during a task requiring the top-down generation of affective states [38].
Anodal tDCS over the right dlPFC either increased or decreased physical and emotional arousal, depending on the assigned objective (to up-regulate vs to down-regulate emotional response, respectively) [39]. (Causal Evidence)
[ Decision-making ]
Integration of Multiple Sources of Information:
The dlPFC is more activated during decisions that require the consideration of multiple sources of information, and it may activate other brain regions during the decision-making process. In comparison, the orbitofrontal cortex (OFC) and ventromedial PFC (vmPFC) are more responsible for making decisions based on the reward and affective values of different options [40].
Personal Moral Dilemma:
BA46 is found to be more activated during decisions regarding personal moral dilemmas compared to impersonal moral dilemmas, and also when compared to other Brodmann areas [41].
[ Cognitive Control ]
(Inappropriate) Response Inhibition:
Excitatory repetitive transcranial magnetic stimulation (rTMS) over the left dlPFC improved response inhibition in the Go/No Go task compared to the sham control group [42]. (Causal Evidence)
Participants in the active rTMS group received one 10 Hz rTMS session per day for one week over the left dlPFC. The active rTMS group showed decreased metabolism in the left dlPFC post rTMS treatment, as indexed by the myo-inositol /creatine complex (MI/Cr) ratio, suggesting decreased energy use, and thus, a less active dlPFC [42].
Cathodal tDCS over the dlPFC increased the likelihood of incorrect impulse response in the cognitive reflection test (CRT) compared to anodal and sham groups, suggesting reduced inhibitory cognitive control [43]. (Causal Evidence)
Solution Evaluation & Recognition:
Anodal tDCS over the left dlPFC enhanced solution recognition (recognizing the correct solution among multiple options) to difficult problems, but not easy problems. Anodal tDCS of the left dlPFC did not enhance solution generation (creative problem-solving), regardless of the difficulty of the verbal insight problem [44].
Anodal tDCS over the right dlPFC improved performance in the creative solution evaluation stage (convergent thinking) but not in the preceding stage of creative solution generation (divergent thinking) for real-life product design problems [45].
Excitation of the dlPFC improves solution evaluation and recognition, but not solution generation. This is aligned with the dlPFC’s role in recruiting and integrating various streams of inputs when making decisions, as evaluating and thus recognizing the optimal solution requires the consideration of multiple sets of information. As well as with the non-involvement of dlPFC in imagination, which primarily involves the hippocampus and the default mode network (more on this later).
[ Cognitive Flexibility ]
Integration of Relevant Novel Information:
Conflict-induced Behavioral Adjustment:
The anterior cingulate cortex (ACC) is responsible for detecting and relaying conflicting information regarding the relevant tasks to the dlPFC. The dlPFC then processes such information and makes necessary neural, and thereby behavioral adjustments such as task- and task-set-switching for goal attainment [48].
6 Hz (theta-band) transcranial alternating current stimulation (tACS) over the dlPFC enhanced conflict-induced behavioral adjustment, as indicated by a reduced Stroop effect [49].
[ Time-processing ]
Right dlPFC & Time Awareness:
Anodal tDCS over the right dlPFC resulted in judging a specific time interval to be longer than it is (e.g. judging the time interval to be 2 seconds when it has only been 1.5 seconds). This effect is only found for time intervals that are in the seconds but not milliseconds range. This effect is found for both the anodal and sham conditions, whereas the opposite effect was found for the cathodal condition [50].
i.e. Time feels slower, potentially due to stimulation of one’s attentional awareness of time (the right dlPFC), and thus, the subjective experience of time is expanded: one second can feel like it is longer because you are more focused on and thereby more immersed in the experiencing of it.
Left dlPFC & Time Accumulator:
A correlational fMRI study suggested that the left dlPFC acts as a “time accumulator,” where it accumulates temporal information as time progresses [51]. This is potentially due to the left dlPFC’s critical involvement in the working memory system, as the tracking of time, or the accumulation of temporal information, is also reliant on remembering how much time has passed (the WM system).
In general, excitatory stimulation of the dlPFC enhanced its associated functions, and inhibitory stimulation impaired its associated functions. The dlPFC is shown to become less active (more inhibited) post excitatory stimulation.
(Note: No literature was found on CB1R activation, by endogenous or exogenous cannabinoids, in the dlPFC.)
[ The Hippocampus (HPC) ]
[ Introduction ]
The hippocampus is a subcortical collection of nuclei in the medial temporal lobes of the cerebral hemispheres and shares extensive connections to the cerebral cortex and the amygdala. Anatomically, it is divided into several regions: the dentate gyrus (DG), CA1, CA2, and CA3 [52].
The hippocampus and parahippocampal regions are considered critical components of the declarative memory system. They are responsible for the formation, organization, storage, and retrieval of conscious memories, which include both episodic and semantic memory [53].
(Declarative Memory: aka explicit memory, refers to memory that can be consciously recalled.)
v.s.
(Non-declarative Memory: aka implicit memory, refers to memory that can be accessed without conscious thought. This includes motor skills/procedural learning, subconscious/perceptual priming, and classical conditioning.)
(CA stands for cornu ammonis.)
(The earlier distinction of hippocampal CA4 is in fact the polymorphic layer of the dentate gyrus, aka the hilus or the hilar region [53].)
[ HPC Connections ] [54]
The Tri-synaptic Loop: together, the [ EC → DG ], [ DG → CA1 ], and [ CA1 → CA3 ] pathways are called the tri-synaptic loop of the HPC.
The Perforant Pathway: The Entorhinal Cortex (Neocortex) → Granule Cells (Dentate Gyrus)
[ EC → DG ]
The perforant pathway serves as the main input stream into the HPC. It consists of neuronal axons that carry processed cortical commands from the entorhinal cortex to the granule cells in the dentate gyrus.
(The Entorhinal Cortex: the main cortical region responsible for integrating sensory and cognitive information for memory processes (consolidation, retrieval, and etc.).)
The Mossy Fiber Pathway: Granule Cells (Dentate Gyrus) → Pyramidal Neurons (PNs) in CA3
[ DG → CA3 ]
The axons of granule cells in the DG are called mossy fibers, and they form large synaptic boutons that strongly excite CA3 PNs. While the DG transforms incoming memory information into distinct neural representations, CA3 PNs further separate these representations (pattern separation) in preparation for memory consolidation in the recurrent pathway within CA3.
The Schaffer Collateral Pathway: Pyramidal Neurons (PNs) in CA3 → Pyramidal Neurons (PNs) in CA1
[ CA3 → CA1 ]
The axons of CA3 PNs are called Schaffer collaterals, and they project onto CA1 PNs. Since CA1 is the output region of the HPC, CA3 → CA1 connections allow CA1 to retrieve memory contents to relay to other brain regions. LTP/LTD along the CA3 → CA1 synapses (Schaffer collaterals) underlie memory formation and learning.
Pathways Formed by CA3 PNs:
The Recurrent Collateral Pathway
PNs within the CA3 region form recurrent connections with each other. These connections are highly plastic and form feedback loops where the neural signals are cycled through the interconnected CA3 PNs multiple times. These recurrent collaterals consolidate activated neural patterns and their associated memory contents (pattern completion) and allow for linking incomplete inputs with the rest of its stored patterns (auto-association).
The Commissural Pathway
The commissural pathway consists of inter-hemispheric connections formed by bilateral CA3 PNs. This pathway facilitates neural communication between the left and right hippocampi (HPCs), enabling the exchange and integration of information across both hemispheres of the brain.
CA2 has distinct cellular and molecular structure compared to CA1 and CA3:
The Output Pathways in CA1
(Dorsal) CA1 → Subiculum → Entorhindal Cortex
This output pathway is primarily involved in spatial memory output for spatial navigation such as spatial episodic memory. Many PNs in the dCA1 region are classified as place cells. These place cells activate when one is in or recognizes a familiar location.
(Subiculum: the subiculum primarily contains PNs and is located at the base of the hippocampus. It serves as a transitional region, connecting CA1 on one end and the entorhinal cortex on the other end.)
(Ventral) CA1 → Medial Prefrontal Cortex, Nucleus Accumbens, and Amygdala
This output pathway primarily relays memory contents involved in emotional/affective and motivated behaviors. The vCA1 PNs project to the medial prefrontal cortex, which is crucial for reward-related executive functions and decision-making; the nucleus accumbens, which is important for reward/punishment processing and motivation; and the amygdala, which plays a key role in emotional processing. Together, these connections help integrate memory contents with emotional and motivational aspects of behavior.
[ CB1R Distribution ]
CB1R is widely expressed throughout all hippocampal regions [57].
Within the hippocampus, CB1R is primarily expressed in the presynaptic nerve terminals of GABAergic interneurons. Using rat hippocampal slices, 96.9% of CCK+ interneurons expressed CB1R, whereas only 4.6% of PV+ interneurons showed CB1R presence. [58, 57]. Specifically, CB1R+/CCK+ interneurons surrounded the perisomatic regions of pyramidal neurons, while PV+ interneurons at these locations showed no CB1R expression [58]. This suggests that CB1R primarily modulates hippocampal GABAergic transmission through inhibiting CCK and GABA release from CCK+ perisomatic basket cells. Thus, CB1R activation on CCK+ interneurons should lead to an increase in not only glutamatergic transmission, but also the fast-acting, inhibitory PV (and GABA) transmission when inhibitory control is needed.
(Recap: PV+ Interneurons: a specific type of inhibitory GABAergic interneurons characterized by the expression of parvalbumin (PV); PV+ interneurons release GABA and PV to form fast-spiking, high-frequency, and precise inhibitory synapses, and these synapses usually target the cell bodies and proximal dendrites (aka the perisomatic regions) of excitatory neurons. Within the hippocampus, PV+ interneurons do not typically express CB1R.)
v.s.
(CCK+ Interneurons: a specific type of inhibitory GABAergic interneurons that release GABA and cholecystokinin (CCK) to form inhibitory synapses. CCK+ interneurons are characterized by asynchronous neurotransmitter release, or the delayed release of CCK and GABA relative to the action potential that triggered it. Hippocampal CCK+ interneurons tend to target the perisomatic regions of hippocampal pyramidal neurons [59].)
CB1R is also found in the presynaptic nerve terminals of hippocampal glutamatergic neurons (i.e. pyramidal neurons in the CA1, 2, and 3 regions, and granule cells in the dentate gyrus), but to a lesser extent [60, 57].
(Pyramidal Neurons: the main output (excitatory) neuron in the cerebral cortex and the hippocampus. These neurons have a pyramid-shaped cell body and a long apical dendrite that extensively branches out towards the cortical surface, enabling them to communicate over long distances with neurons in other brain regions.)
As in other brain regions, CB1R is also found in hippocampal astrocytes [61], as well as the mitochondria of various hippocampal cell types, including interneurons, principal pyramidal neurons, and astrocytes [57, 62].
[ Functions ]
[ Neurogenesis ]: the production of new brain cells (aka neurons); the dentate gyrus in the hippocampus is one of the regions that harbor multipotent neural progenitor (NP) cells, which are the precursor cells needed for adult neurogenesis [63].
CB1R activation in NP cells promotes NP cell proliferation and neurosphere generation [64].
[ Memory ]: the neural substrate underlying memory, and thus, learning, is the changing of synaptic plasticity, or neuroplasticity of (hippocampal) neurons through either long-term potentiation (LTP) or long-term depression (LTD). For example, updating a previously made association, such as re-associating a familiar location with a novel object, induces LTD [65]. Specifically, the hippocampus acts as a long-term storage system for recently acquired events [66]. The ECS participates in such processes through facilitating (long-term) changes in GABAergic release, and thus, LTP or LTD in neuronal synapses [57]. The hippocampus is also involved in working memory. Specifically, the holding of multiple information/items [67], and deactivation of the hippocampus during working memory encoding predicts failure of long-term encoding of such information [68].
In general, normal hippocampal CB1R functioning through eCB signaling is required for various types of memory. Hippocampal CB1R activation by exogenous cannabinoids, or its absence through genetic deletion, tends to impair LTP and/or LTD, leading to disruptions in memory processes (e.g. consolidation).
Hippocampal astroglial CB1R modulates both LTP and LTD at the CA3-CA1 synapses, which govern novel object recognition (NOR) episodic-like memory encoding and (spatial) working memory:
Astroglial CB1R activation is required for the initial consolidation stage of long-term NOR memory because its activation is needed for the release of D-serine, which activates the excitatory NMDA receptor (NMDAR) of the postsynaptic neuron to induce LTP [69]:
Deletion of hippocampal astroglial CB1R (mutant mice) impaired long-term NOR memory and decreased LTP compared to mice with normal CB1R expression (wild-type or WT).
The reversal of astroglial CB1R in mutant mice completely abolished these memory impairment effects, whereas blockade of NMDAR in WT mice fully prevented LTP induction at the CA3-CA1 synapses.
Injection of D-serine immediately (1hr) after novel object acquisition (i.e. the training phase) in mutant mice fully restored LTP and NOR impairment. This effect is absent when the injection happened immediately before training, suggesting that D-serine-induced LTP at CA3-CA1 is only relevant in the initial phase of NOR (episodic) memory consolidation.
Astroglial CB1R activation by exogenous CB1R agonists (HU210 and Δ9-THC) can also impair (spatial) working memory and induce LTD at CA3-CA1 synapses but not the perforant pathway. This is due to the activation of the NMDAR, which induces either endocytosis or the internalization of AMPAR from the postsynaptic membrane [20].
The exogenous cannabinoids-induced spatial working memory impairment and LTD were eliminated in mice lacking hippocampal astroglial CB1R, but not in those lacking hippocampal GABAergic or glutamatergic CB1R.
Therefore, hippocampal astroglial CB1R activation modulates both LTP and LTD at the CA3-CA1 synapses through activating NMDAR at the postsynaptic neuron, which then can induce either LTP or the internalization of AMPAR, which leads to LTD.
Hippocampal GABAergic CB1R activation modulates one’s ability to distinguish memory contents between trials [70].
The asynchronous inhibition induced by CCK+ interneurons may play a critical role in the segregation of distinct memory contents by allowing the consolidation of one memory content into their respective pyramidal neurons before inhibiting their activities (delayed inhibition); and before moving on to the encoding of the next memory trial (and thus, the activation of a distinct set of pyramidal neurons), thereby enhancing the fidelity of memory encoding. {Research Question}
Hippocampal GABAergic CB1R (mostly on CCK+ interneurons) functions as a circuit breaker to prevent excessive glutamate release, and thus, excitotoxicity [71].
Mice with GABAergic CB1R deletion in the forebrain, which includes the hippocampus, increased age-related loss of principal neurons in the hippocampus, but not in other brain regions; and exacerbated neuroinflammation marked by higher astrocytes densities in the CA1 and CA3 regions, activated microglial cells (with similar numbers of resting microglial cells) in the CA1 region, and age-related expression of pro-inflammatory cytokines (IL-6 and TNF but not IL-1β) compared to their WT littermates [71].
CB1R-deficit mice showed decreased neurogenesis compared to normal mice during juvenile/adolescence (2-mo-old mice), but this difference disappears as age progresses (5-mo- and 12-mo-old mice)[71], suggesting that CB1R plays a crucial role in inducing neurogenesis during adolescence.
(Forebrain: including the cerebrum and all subcortical structures other than the cerebellum and the brain stem.)
Hippocampal GABAergic CB1R activation by THC leads to increased NMDAR-mediated excitatory transmission. This CB1R activation temporarily enhanced the protein synthesis machinery necessary for synaptic plasticity, and thereby memory consolidation through the mammalian target of the rapamycin (mTOR)/p70S6K pathway. This (abnormal) increase in protein synthesis is correlated with the amnesic effect of THC, as both p70S6K phosphorylation and THC-induced memory impairment are reduced in mice with GABAergic CB1R knockout (i.e. deletion) or NMDAR blockade [72].
CB1R is also expressed in hippocampal dopamine 1 receptor (D1R)-positive cells (D1R is found on hippocampal GABAergic interneurons and glutamatergic principal neurons). CB1R on GABAergic interneurons that co-express D1R is required for the late consolidation stage of NOR (episodic) memory [66].
Mutant mice with deletion of CB1R in the hippocampal D1R+ cells showed impaired NOR memory 24hrs (long-term), but not 3hrs (short-term) after training, suggesting impairment to the late consolidation stage of long-term NOR (episodic) memory.
At the CA3-CA1 synapses, mutant mice showed similar high-frequency stimulation (HFS)-induced LTP to their WT littermates when the HFS was applied without NOR training. However, when HFS was applied after NOR training, WT mice showed enhanced LTP whereas mutant mice lacked this learning-induced enhancement in LTP.
In the hippocampus, D1R is found in a small subset of GABAergic interneurons that have relatively low levels of CB1R. The co-expression of D1R on this small subset of GABAergic interneurons was absent in mutant mice. Thus, the remaining hippocampal D1R that were being tested in this study were mainly on glutamatergic principal neurons.
The NOR impairment is likely due to excessive transmission of D1R+ cells without CB1R-mediated inhibition:
Injection of hippocampal D1R+ cell inhibitor 1hr after training fully abolished the NOR impairment.
GABAa receptor antagonist, but not glutamatergic NMDAR and AMPAR antagonists, injected 1hr after training fully rescued the NOR impairment and induced training-related LTP in mutant mice (GABAa receptor did not further enhance training-induced LTP in WT mice).
D1R partial inhibitor injected after training eliminated the difference in training-induced LTP in mutant compared to WT mice, and fully eliminated the memory impairment effect in mutant mice.
Together, these findings suggest that deletion of CB1R from hippocampal D1R+ cells resulted in:
Excessive GABA transmission in local inhibitory interneurons without CB1R-mediated inhibition.
Excessive D1R activation on principal neurons without CB1R-mediated inhibition. In mutant mice, D1R is primarily located on principal neurons since D1R co-expression on GABAergic interneurons is absent.
Diminished learning-induced LTP, likely due to excessive GABA transmission in local inhibitory interneurons and/or excessive activation of D1R in local principal neurons.
Impaired the late consolidation stage of long-term NOR (episodic) memory, likely due to a lack of LTP at the CA3-CA1 synapses.
Exogenous cannabinoids-induced memory impairment is potentially due to excessive activation of CB1R (over-excitation), lack of CB1R availability for eCB signaling, or both. {Research Question}
[ Incidental Associations ]: aka mediated or inferred learning, incidental association is the meaningful association of relatively unrelated stimuli with a positive or negative outcome, depending on the context in which they are formed. These relatively unconscious but reasonable associations can then be used to guide decision-making in the future.
Hippocampal CB1R activation is required to form incidental associations, likely due to its role in controlling inhibitory transmission [73, 74].
Hippocampal CB1R blockade during the preconditioning stage of mediated learning, where the association between two relatively unrelated stimuli was formed, impaired mediated learning, potentially due to excessive inhibitory transmission [73].
Re-expressing hippocampal CB1R in mice lacking CB1R globally (i.e. in the entire brain) fully rescued their mediated learning ability, suggesting that CB1R in the hippocampus specifically mediates incidental associations [73].
[ Creativity & Imagination ]: Creativity is often broken down into two aspects: divergent thinking (idea generation) and convergent thinking (idea evaluation). Divergent thinking relies on episodic memory, a primary function of the hippocampus, as it often requires imagination — the picturing (episodic simulation) of the future. Convergent thinking is found to activate not only the hippocampus and the default mode network, but also the frontal brain regions involved in cognitive control, including the dlPFC [75, 76].
The Default Mode Network: connection shared among the medial prefrontal cortex, posterior cingulate cortex, bilateral inferior parietal lobes, and medial temporal lobes (encompasses the hippocampus and amygdala) [75].
The default mode network activates “by default” during relaxation when one is not engaging in any cognitive task. This spontaneous thinking or mind-wandering state often involves recalling past experiences and imagining future scenarios.
Constructive Episodic Simulation Theory: both episodic memory and episodic simulation of the future require the flexible reconstruction of (past) episodic details, such as people, places, and events.
Episodic retrieval, future simulation, and idea generation all activated the bilateral hippocampi, along with the inferior frontal gyrus (involved in memory retrieval) and middle occipital gyrus (involved in mental imagery generation) [77].
The default mode network was activated during both the idea generation and idea evaluation stages during a creative task. However, the idea evaluation stage recruited the executive control brain regions in addition to the default mode network [76, 78].
The functional connectivity (i.e. communication) between the default, executive, and salience networks (the salience network governs the ability to switch between default and executive networks) is strongly and positively correlated with creativity [79].
Damage to the hippocampus impairs one’s ability to not only recalling the past, but also imagining the future [75]. On the other hand, episodic induction (prompting the subject to recall an episodic memory in a high degree of detail) strongly activated the default mode network, specifically, the left anterior hippocampus, and improved divergent thinking (the number and variety of ideas generated) [76].
Memory contents in the hippocampus are stored and represented through sparse encoding. CB1R activation in the hippocampus increases the overall excitation of the hippocampus, potentially allowing access to more (episodic) memory contents simultaneously, thereby increasing the combinatorial possibilities of the activated episodic details, leading to enhanced imagination. {Research Question}
[ The Amygdala ]
[ Introduction ]
The amygdala is a subcortical collection of nuclei located in the medial temporal lobes of the cerebral hemispheres, positioned in front of the hippocampus. It plays a superordinate role in emotional processing by evaluating sensory information and assigning emotional values accordingly [80].
[ CB1R Distribution ]
CB1R is found to be highly expressed in the basolateral amygdala (BLA), which consists of the lateral and basal nuclei. CB1R is very low or not present in the central and medial nuclei. In the BLA, CB1R is primarily expressed in the presynaptic axons of GABAergic large CCK+ interneurons [81, 82].
The Basolateral Amygdala (BLA)
The BLA contains mainly excitatory principal neurons and exhibits a cortical-like neuronal composition [83]. The BLA is a region associated with mediating anxiety and fearful/aversive memories. Increased neuronal excitability and dendritic hypertrophy in the BLA are associated with anxiety-like behavior, and BLA activation is found during the formation, expression, and extinction of fearful memories [84].
The Lateral Nucleus of the Amygdala (LA)
The lateral nucleus (LA) is the input region and the origin of intra-amygdala projections. It receives external sensory inputs from the thalamus, associative cortices, and brainstem. These inputs are then relayed to the basal nucleus as well as other amygdala regions, including the central and accessory basal nuclei [54, 83].
The Basal Nucleus of the Amygdala (BA)
The basal nucleus (BA) integrates the LA-incoming sensory information with its received top-down perceptual inputs from the mPFC, contextual inputs from the vHPC, and neuromodulatory inputs from regions such as the VTA and basal forebrain. The BA then sends this integrated information back to the vHPC and mPFC, as well as to other amygdala regions [83].
(Neuromodulatory Inputs: signals from neuromodulators such as dopamine, serotonin, acetylcholine, and norepinephrine.)
[Functions]
Exogenous CB1R activation in the amygdala increased the excitability of amygdala principal neurons (PNs):
Activation of amygdala CB1R by exogenous agonists WIN 55,212–2 and CP 55,940 reduced both GABAa receptor-mediated and spontaneous inhibitory postsynaptic currents (IPSCs) in BLA principal neurons (PNs), suggesting less inhibitory transmission, and thus, more excited PNs. CB1R activation did not affect action potential-independent IPSCs, nor did it affect any IPSCs in the central and medial nuclei where CB1R is not expressed [82].
Exogenous CB1R activation in the LA is also found to induce an overall reduction in PN excitability:
Contrarily, CB1R activation in LA by WIN55,212-2, while decreasing both glutamatergic and GABAergic synaptic transmission, induced an overall decrease in neuronal excitability [90].
CB1R activation by exogenous agonists can both increase and decrease the overall excitability of PNs in the BLA/LA.
[ Stress & Anxiety ]
Exposure to stress increases neuronal excitability in the BLA, which then activates the hypothalamic-pituitary-adrenal (HPA) axis and induces anxiety-like behavior [84].
Amygdala CB1R-knock out induced anxiety-like behavior (disrupted night sleep, agitated psychomotor activity in new environments, and decreased social desire) and increased plasma cortisol levels in marmosets (monkeys) [91].
CB1R-KO mice showed increased aggressive- and depressive-like behaviors in a chronic unpredictable mild stress environment, likely due to having a less effective emotional regulatory system (the ECS on the BLA on the HPA axis) [92].
CCK in the amygdala is anxiogenic (anxiety-inducing), and thus, inhibition of CCK with CB1R activation is anxiolytic (anxiety-reducing) [93].
Thus, CB1R plays an essential and positive role in regulating stress and anxiety by regulating the excitability of the BLA, and subsequently, the HPA axis.
[ Fear/Aversive Memory Extinction ]
During fear extinction, BLA showed elevated eCB levels [94], and eCB-mediated CB1R activation can induce both LTP and LTD to facilitate neuroplasticity changes underlying the extinction of aversive memories.
CB1R activation is required for fear/aversive memory extinction, but not for reward-associated memory extinction:
CB1R-KO mice showed strong short-term and long-term deficits in the extinction of aversive memory (auditory fear-conditioning stimulus), with no change in the acquisition and consolidation of fearful memory [94].
CB1R antagonist in WT mice also showed impaired fear extinction [94].
Mice with systematic deletion of CB1R (CB1R-) and WT mice showed similar decline in performance when the food reward was removed from a previously conditioned food task, suggesting that CB1R is not required for the extinction of positively-reinforced memory [95]:
CB1R- mice showed less motivation to participate in task with food reward compared to WT mice. CB1R- mice eventually reached the same performance level as WT mice with increased food restrictions.
BLA CB1R activation reduces fear response:
BLA CB1R activation by WIN 55,212-2 reduced fear-potentiated startle in rats in a conditioned fear paradigm [96], likely due to increased GABA transmission.
WIN 55,212-2 injection in the amygdala after revisiting fearful memory prevented the re-consolidation of said memory, proxied with fear-potentiated startle. The prevention of fear re-consolidation is stronger with higher dose of WIN 55,212-2, and is blocked by CB1R antagonist AM251. CB1R activation showed no effect in fearful memory re-consolidation when the specific aversive memory was not reactivated prior to the injection of CB1R agonist [97]:
WIN-treated rats showed no shock-induced reinstatement of fear and no spontaneous recovery of fear, without amygdala damage or changes to baseline startle/shock reactivity. This suggests that CB1R activation by exogenous agonists either impairs aversive memory processes or facilitates the abolishment of non-life-threatening fears by inhibiting their re-consolidation.
In summary, CB1R modulates neuronal synaptic plasticity and BLA is a key region for fear extinction. The neural activity of PNs in the BLA translates to the behavior of various fear responses. CB1R-mediated neuroplasticity changes the behavioral response to fear-associated stimuli and is the principal neural mechanism that underlies fear extinction.
Notable Neural Pathways
[ mPFC - BLA - HPC ]: Emotional Association Memory & Learning
mPFC - BLA: the mPFC is the primary top-down control region for this pathway and shares extensive bi-directional connections with the BLA. These connections allow for the determination of the emotional significance of sensory stimuli that activate the BLA (i.e. emotionally salient or relevant stimuli), and of the appropriate emotional responses.
LTP/LTP along the mPFC - BLA neuronal connections is the neural correlate of exposure to emotionally salient contexts, and of the subsequent contextual memory and learning [98]:
Both systematic and within-circuit CB1R blockade by inverse agonist AM251 before conditioning (i.e. the exposure to emotionally salient stimuli) inhibited LTP along the mPFC - BLA pathway and the subsequent behavioral acquisition of emotional (fear) associative memory.
mPFC CB1R activation by exogenous agonist WIN 55,212-2 elicited behavioral response to normally below-threshold (aversive) stimulus [99]:
Systematic CB1R activation potentiated neural responses (increased neuronal firing frequency and bursting activity) in the mPFC to previously conditioned emotionally triggering cues, while CB1R antagonist prevented such neural responses.
mPFC - HPC: the mPFC receives neuronal projections from the HPC [54]. Thereby, the mPFC recruits HPC memory contents to decide the emotional relevance of a given context.
BLA - HPC/mPFC: the more BLA is activated the stronger the memory consolidation in the hippocampus and/or mPFC (stronger BLA activation indicates higher emotional valence of a given stimulus, and thus, stronger encoding (mPFC) and better consolidation of this (episodic) memory (HPC)).
Inhibiting the BLA with muscimol before conditioning prevented learning-associated neuronal activities (bursting) in BLA-responsive mPFC neurons without affecting baseline neural activities. When co-administered with WIN 55,212-2, muscimol pre-treatment also blocked the potentiation effect of this CB1R agonist [99], as CB1R-mediated enhancement of synaptic transmission was no longer available.
Inhibiting the BLA with GABAa receptor agonist muscimol decreased the consolidation of (fear-related) contextual memory in the hippocampus [54].
CB1R activation in the BLA is more likely to excite the BLA due to its primary localization on the presynaptic axon terminals of GABAergic CCK+ interneurons. {Research Question}
CB1R activation (by exogenous agonist) affects the mPFC-BLA-HPC pathway in a way that promotes the detection of normally sub-threshold BLA-activating stimuli by eliciting stronger neural responses in the association region (mPFC) to previously conditioned cues. This effect is not restricted to fear-associated cues, as the BLA also processes rewarding cues [100]. CB1R blockade diminishes the neural correlates of emotional associative learning, suggesting its critical role in the encoding, acquisition, and consolidation of emotional associative memories, and consequently, learning.
[ The Mesocorticolimbic System ]: Motivation & Reward/Punishment Processing
The mesocorticolimbic system consists of DAergic neurons in the VTA that project to the NAc, the PFC, the amygdala, and the HPC, as well as other neuronal connections shared among these regions. Neurons in this pathway primarily release dopamine (DA) to communicate with one another. Thus, they are considered dopaminergic (DAergic) neurons.
DAergic neurons exhibit two types of firing patterns: tonic and phasic. Tonic firing refers to the spontaneous yet consistent low-frequency single spikes of DA release that maintain baseline DA levels. Phasic firing involves rapid high-frequency multi-spikes (”bursts”) of DA release in response to significant stimuli. While phasic DA release is necessary for forming and updating the associations between predictive cues and the subsequent rewards or punishments (long-term emotional association memories), tonic DA determines one’s motivation to act upon such cues [101].
VTA - NAc: the mesolimbic neurons are DAergic neurons in the VTA that send one-directional projections to the NAc. Both rewarding and aversive stimuli trigger phasic DA release in the NAc [102]. Thus, NAc DA release regulates both the pursuit of reward and the avoidance of punishment.
VTA - mPFC: the mesocortical neurons are DAergic neurons in the VTA that share reciprocal connections with the mPFC.
DA release in the mPFC mediates the cognitive control processes in motivated behaviors. Specifically, in filtering sensory inputs, shifting WM focus towards the most relevant reward/punishment-associated stimuli, and relaying final decisions to motor regions to initiate motivated actions [104].
For mice with established cocaine-conditioned place preference, the presentation of cocaine-associated cues increased DA level in the PFC but not NAc. On the other hand, when mice were placed in the apparatus between two connected chambers — one potentially with cocaine — NAc DA level increased as exploration of the chambers required motivation, which was mediated by NAc DA transmission [105].
mPFC DA transmission tends to bias motor commands toward responding to stimuli that is associated with potential aversive outcomes [106].
vHPC - NAc Shell: the vHPC sends direct excitatory projections to the NAc shell, where it innervates the dendrites of DAergic neurons projecting from the VTA [85]. Therefore, vHPC modulates DA release in the NAc shell through exciting VTA DAergic neurons, and as a consequence, affects reward processing of external stimuli:
[ The Nigrostriatal Pathway ]: Motivated Movements
The nigrostriatal pathway, along with the mesolimbic and mesocortical pathways (collectively known as the mesocorticolimbic system), are three of the four major DAergic pathways in the brain. The nigrostriatal pathway consists of DAergic neurons located in the substantia nigra pars compacta (SNc) that project to the putamen and the caudate nucleus of the striatum (dorsal striatum). DA transmission in this pathway mediates voluntary movements including bodily posture [107, 108].
(Substantia Nigra (SN): the SN is a midbrain structure. It is divided into two parts: the substantia nigra pars compacta (SNc) and the substantia nigra substantia nigra pars reticulata (SNr). The SNc is primarily made up of DAergic neurons while the SNr mainly consists of GABAergic neurons.)
(Striatum: the striatum is a subcortical structure in the forebrain. It is divided into three parts: the putamen, caudate nucleus, and nucleus accumbens.)
Dopamine is considered a neuromodulator as it modulates the release of other neurotransmitters (e.g. serotonin, norepinephrine, glutamate, and GABA). Although CB1R is not expressed on midbrain DAergic neurons, it is highly expressed in regions receiving these DAergic projections, and on GABAergic interneurons that innervate these DAergic neurons. Thus, the ECS plays a superordinate regulatory role over the DAergic systems, where it can either enhance or inhibit the release of DA through indirect mechanisms [107, 109, 110].
Other Notable Regions
CB1R is highly expressed in brain regions that process bodily sensations.
[ The Solitary Nucleus ] [111]
Aka the nucleus of the solitary tract (NST) or the nucleus solitarius. The NST is located in the medulla oblongata, which is part of the brainstem [112]:
Processes visceral sensory information as it receives all visceral afferents.
Regulates internal homeostasis through its extensive connections with the cardiovascular, respiratory, and gastrointestinal systems.
The NST receives sensory information from 3 out of 12 cranial nerves (CN). Specifically, CN 7, 9, and 10: the facial nerve (CN VII), glossopharyngeal nerve (CN IX), and vagus nerve (CN X).
The Facial Nerve (CN 7):
Receives taste inputs from the front 2/3 of the tongue
Sends motor commands to muscles that control facial movement and expression, and to the gland that produces tears (lacrimal gland)
The Glossopharyngeal Nerve (CN 9):
Receives taste inputs from the back 1/3 of the tongue
Contains general visceral afferent (GVA) fibers that carry sensory inputs from the carotid sinus and carotid body (neck)
Contains efferent (outward) branchial motor fibers that control the movement of the pharynx (throat) and larynx (voice box)
The Vagus Nerve (CN 10):
Contains about 80% afferent fibers and 20% efferent fibers, and is the principal nerve regulating interoceptive awareness [113, 114]
Relays sensory information in the gut-brain axis [115]
Processes viscerosensory signals along with visual/auditory/olfactory patterns [116]
Receives taste inputs from the epiglottis
Contains general visceral afferent (GVA) fibers
[ The Dorsal Horn of the Spinal Cord ] [117]
The dorsal horn is a specific division of the grey matter in the spinal cord. It is present at all levels throughout the spinal cord.
Consists of cell bodies that receive and process somatosensory information (touch, pain, and temperature perception) from the skin, muscles, and viscera (internal organs) of the body.
In short, the solitary nucleus is the main relay and processing center for visceral sensory information as it receives all visceral afferents. The dorsal horn of the spinal cord is the region within the CNS that receives the first relay of somatosensory information from peripheral sensory neurons.
CB2R
CB2R is the primary CBR in the PNS while CB1R is the primary CBR in the CNS. CB2R is abundantly expressed in the immune system including hematopoietic cells and immune cells. Additionally, CB2Rs are also found in peripheral tissues such as the cardiovascular system, reproductive system, GI tract, adipose tissue, spleen, liver, and bone [9]. CB2R is found to be upregulated in the CNS under pathological conditions, such as neuroinflammation [118], as its activation modulates immune and anti-inflammatory responses.
Other CBRs
Other than the most well-studies CB1R and CB2R, eCBs and exogenous cannabinoids also interact with other receptors [1]:
Other G protein-coupled receptors (GPCRs)
GPR55, GPR18, GPR3, GPR6, GPR12
Transient Receptor Potential (TRP) Channels
TRP vanilloids (TRPVs): TRPV1, TRPV2, TRPV3, TRPV4
TRP ankyrin (TRPAs): TRPA1
TRP M member (TRPMs): TRPM8
Peroxisome Proliferator-activated Receptors (PPARs)
PPAR2, PPARγ
Monoamine Transporters
Norepinephrine Transporter (NET)
Dopamine Transporter (DAT)
Serotonin (5-HT) Receptors
Serotonin 1A (5-HT1A) Receptor
Serotonin 3 (5-HT3) Receptor [7]
Nicotinic Acetylcholine Receptors (nAChR)
α7 nAChR [7]
Glycine Receptors (GlyR)
GlyR α1, GlyR α3
(Note: this list is not comprehensive.)
[2] The Endocannabinoids (eCBs)
The endocannabinoids (eCBs) are lipid-based neurotransmitters that bind to and activate the cannabinoid receptors. They are produced on-demand by the postsynaptic neuron and travel backward to bind to the cannabinoid receptor on the presynaptic neuron (aka retrograde signaling) [9]. There is also evidence for anterograde eCB-signaling [119].
The two most well-known and well-studied eCBs are AEA (N-arachidonoylethanolamine), and 2-AG (2-arachidonoylglycerol). Both are arachidonic acid derivatives (arachidonic acid is a type of polyunsaturated fatty acid, specifically, an omega-6 fatty acid). Their precursors (glycerophospholipids) are present in the lipid membrane of the cell/neuron and can be rapidly produced in one or two enzymatic steps [9, 120]. The eCBs are found in the circulation of blood throughout the body, and their concentration is dynamic. They are found in all organs/tissues/bodily fluids examined so far [121].
Both AEA and 2-AG bind to CB1R, CB2R, and/or other cannabinoid receptors to exert their various effects. The specific physiological processes and the subsequent behavior outcomes they trigger depend on the brain region in which the cannabinoid receptor activation takes place, as extensively discussed above.
AEA
AEA, aka Anadamide (ananda means internal bliss, so anadamide means amide of the internal bliss) shares many properties with THC and has been referred to as “an endogenous marijuana-like substance self-delivered by the brain” [122]. The brain has the highest AEA concentration among all other tissues [123].
While AEA is a partial agonist at both CB1R and CB2R, it has a higher binding affinity to both receptors compared to 2-AG [124]. AEA also binds to other CBRs such as the TRPV1 channel and PPARs [123].
(Partial Agonist: a molecule/ligand that activates a receptor but produces a sub-maximal response compared to a full agonist.)
v.s.
(Full Agonist: a molecule/ligand that fully activates a receptor, inducing the maximum possible response the receptor can produce.)
(Binding Affinity: the strength of interaction between a molecule/ligand and its binding partner such as a receptor, often measured by how tightly and how long the two bind to each other.)
AEA increases optimism:
Increasing AEA by inhibiting its degrading enzyme FAAH biased the rats toward a more optimistic interpretation of the ambiguous cue in the ambiguous-cue interpretation paradigm. This effect was eliminated with either CB1R or CB2R blockade, suggesting the involvement of both receptors in mediating the optimism effect of AEA [125].
(The Ambiguous-Cue Interpretation (ACI) Paradigm: The ACI is used to test the valence/hedonic tone of cognitive judgment bias (optimistic/pessimistic) in animals. First, the researchers train the animals to associate a specific tone with pressing a specific lever (the positive lever) to receive a reward such as food, and to associate a different tone with a different lever (the negative lever) to avoid a punishment such as a mild electric foot shock. The training phase ends when the animals can consistently reach a specific discrimination ratio. Then, the animals are presented with a tone that is in the middle of the positive and negative tones. Their choice of pressed lever therefore indicates their cognitive judgment of the ambiguous cue.)
Humans that carry the FAAH 385A allele (about 38% of European descent [126]) have destabilized FAAH protein, which leads to higher levels of circulating AEA:
2-AG
2-AG is a full agonist of both CB1R and CB2R but with a lower binding affinity than AEA. 2-AG is the most abundant eCB in the body, and the brain has the most 2-AG compared to other tissues. Its concentration is significantly higher than that of AEA within the same tissue [129, 130]. For example, 2-AG is approximately 1000 times more abundant than AEA in the brains of nonhuman primates [124]. 2-AG is not known to act on TRPV1 [123].
2-AG vs AEA
It is said that 2-AG mainly acts as a phasic eCB, where its release is rapid, transient, and in larger amounts to mediate synaptic plasticity such as DSI/DSE (short-term) or LTP/LTD (long-term). However, AEA can also mediate synaptic plasticity. Tonic eCB signaling, which is found to modulate physiological processes such as mood, sleep, pain, and feeding behaviors, depending on the specific location of CBR activation, is found to be mediated by both 2-AG and AEA [131].
Since 2-AG selectively binds to CBRs while AEA also interacts with other classes of receptors, 2-AG has been proposed to be the true ligand for the ECS [124]. Side note, it requires the pharmacological inhibition of the degradation enzymes of both AEA and 2-AG to produce similar behavioral effects to those of THC [132], suggesting that cannabis affects the ECS in a way that is synonymous with the combined actions of 2-AG and AEA.
Perhaps AEA-mediated synaptic plasticity serves as the neural substrate specifically for spiritually enlightening experiences, while 2-AG mediates the regulatory role of the ECS. Just like how serotonin overall induces a sense of calm and contentment, dopamine a sense of motivation and reward, and acetylcholine a sense of focus. AEA might specifically induce spiritual enlightenment kind of pleasure (running/runner’s high, singing, and exercising all increase AEA; social play increases AEA as well). {Research Question}
Other eCBs
Although AEA and 2-AG have been the most studied eCBs — defined as molecules that can interact with the CBRs including and extending beyond CB1R and CB2R — there are other signaling lipids that can also interact with the CBRs.
For example, the NAEs (N-acylethanolamines), which include PEA (*N-*palmitoylethanolamine), OEA (N-oleoylethanolamine), DHEA or synaptamide (N-docosahexaenoylethanolamine), AEA or anadamide (N-arachidonoylethanolamine), and their oxygenated metabolites can all interact with the CBRs [133].
[3] The Endocannabinoids Metabolism Enzymes
Endocannabinoid metabolism enzymes include synthesizing enzymes and degrading enzymes for the eCBs.
Synthesis
AEA is catalyzed from NAPE (N-arachidonoyl-phosphatidylethanolamine) by NAPE-PLD (NAPE-specific phospholipase D), or via other routes not involving NAPE-PLD.
NAPE is produced when N-acyltransferase (NAT) catalyzes phosphatidylethanolamine (PE), a released phospholipid precursor from the plasma membrane [9, 123].
2-AG is synthesized from DAG (diacylglycerol) by DAG lipase alpha or DAG lipase beta; most, if not all, 2-AG that mediates adult brain synaptic transmission is generated by DAGL alpha.
DAG is produced from phosphoinositides by phospholipase C [9, 1].
The rate-limiting and Ca^2+-sensitive step in the production of AEA and 2-AG is the formation of their membrane phospholipid precursors NAPE and DAG, respectively [9].
Degradation
Once eCBs are released into the extracellular space, they are taken into the cells fairly rapidly, and are then degraded intracellularly through hydrolysis and/or oxidation [9, 123].
AEA is primarily hydrolyzed by FAAH (fatty acid amide hydrolase) into free arachidonic acid and ethanolamine. Whereas 2-AG is mainly hydrolyzed by MAGL (monoacylglycerol lipase) into arachidonic acid and glycerol. Cyclooxygenase-2 and some other lipoxygenases are involved in the oxidation of AEA and 2-AG [9].
FABPs (fatty acid-binding proteins) are intracellular proteins that transport AEA and 2-AG to their respective degradation enzyme to be metabolized. FABPs are found to also be the carriers for THC and CBD during their degradation process [123, 134].
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