**2. Discovery of an endogenous cannabinoid system**

The history leading up to the discovery of the "endocannabinoid (eCB) system" is an interesting one, sprouting from a decades- long quest for the active constituents of the marijuana plant, *Cannabis sativa*. Though the cannabis plant has long been used for a variety of purposes dating back more than 4000 years (O'Shaughnessy 1842; Mechoulam and Hanus 2000), only recently was it found that delta 9- tetrahydrocannabinol (Δ9-THC) was the ingredient responsible for the psychotropic effects associated and exploited with its use (Mechoulam and Gaoni 1965).

One of the original and most ancient uses of the *Cannabis sativa* plant was to induce a trancelike state, often an essential component to the elaborate religious rites in ancient cultures ranging from the Chinese, to the Ayurvedic Indians, to the Persians and Greeks (O'Shaughnessy 1842; Aldrich 2006). Herodotus referred to the use of the hemp plant by the

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 101

capable of binding to both the original CB1R, and to this novel CB2R receptor [(Mechoulam,

Fig. 1. **A Historical timeline of the eCB system.** Illustrated above are the major developments

leading up to the discovery of an endogenous cannabinoid system, with the pivotal discoveries of the cannabinoid receptors, CB1R and CB2R, and the ligands AEA & 2-AG. Partly summarized from (Mechoulam and Hanus 2000). Receptors made with motifolio.com©.

Ben-Shabat et al. 1995; Sugiura, Kondo et al. 1995) and see **Figure 1** for a timeline].

Scythians as incense in funeral rites, and also described the use of the hemp plant by the Phoenicians to make 'cordage' for building bridges (Herodotus 1824). The plant was extensively cultivated for its fiber which was used to make fabric for ship sails and clothing, but was also used for food, cooking oil, as a lubricant, and as an analgesic (Grinspoon 1993).

The earliest work to find the active ingredient began in the late 19th century after reports from Dr. O'Shaugnessy during his travels in India. In the true spirit of a responsible clinical researcher, before testing on humans, Dr. O'Shaugnessy described the use of hemp on various animals, a practice not standard for physicians at his time. Based on his findings, he believed that certain patients could benefit from the use of cannabis extracts (O'Shaughnessy 1842). His case studies described the use of the drug in humans for rheumatism, hydrophobia, cholera, tetanus, and infantile convulsions. He cautioned, however, of the "delirium occasioned by continued Hemp inebriation," which continues to be a great -but not insurmountable- obstacle for modern pharmacologists synthesizing drugs targeting the endocannabinoid system. He detailed the effects of cannabis preparations for a variety of ailments in a lecture given to the Medical College of Calcutta in 1839. Based on his work, a renewed interest in active cannabis extracts led to scientific inquiry in Europe and the United States, but an active component was not isolated mostly due to lack of effective techniques available at the time. It was not until 1965 that the major psychoactive constituent Δ9-THC by Mechoulam's group (Mechoulam and Gaoni 1965). By the 1970s, many phytocannabinoids were characterized, and it was determined that they were lipid derivatives. Because of the lipophilic nature of these compounds, their mechanism of action was thought to be mediated by their ability to adhere to cellular membranes, much like the proposed mechanism of anesthetic action (Paton 1975). The isolation of Δ9-THC was a key breakthrough in the discovery of an endogenous cannabinoid system because it allowed for the unexpected identification of a highly specific binding site in the body (Devane, Dysarz et al. 1988). This binding site was isolated and cloned in 1990, from both rat and human tissues (Matsuda, Lolait et al. 1990; Gerard, Mollereau et al. 1991) and was named the cannabinoid 1 receptor (CB1R).

Since it did not seem logical that the body would invest energy in the synthesis of receptors that specifically bind the constituents of this one plant, scientists began looking for compounds produced by the body that could also bind to CB1R. Binding studies with known neurotransmitters and hormones proved to be unfruitful, indicating that a unique ligand was utilizing this newly discovered CB1R. By using a highly specific probe for CB1R labeled with tritium (Devane, Breuer et al. 1992), competitive binding studies in pig brain fractions indicated the presence of endogenous compounds with cannabimimetic activity. Chromatography, nuclear magnetic resonance and mass spectrometry were used to identify arachidonoylethanolamide (Devane, Hanus et al. 1992). An amide group in this newly discovered compound and the historically acknowledged effect of cannabis use, led to the witty alternate name for the very first endocannabinoid 'anandamide', deriving from the Sanskrit word for 'bliss' (Devane, Hanus et al. 1992; Mechoulam 2000). Not only did anandamide work like Δ9-THC in binding assays, it also mimicked its effects on motor functions, sedation and pain relief (Mechoulam 2000).

In 1993, shortly after the discovery of anandamide, another cannabinoid receptor was found and cloned from the periphery (rat spleen), and identified mostly on immune cells (Munro, Thomas et al. 1993). It was referred to as the CB2 receptor (CB2R). Two groups, made the separate discovery of another endocannabinoid, 2-arachidonoyl glycerol (2-AG), that was

Scythians as incense in funeral rites, and also described the use of the hemp plant by the Phoenicians to make 'cordage' for building bridges (Herodotus 1824). The plant was extensively cultivated for its fiber which was used to make fabric for ship sails and clothing, but was also used for food, cooking oil, as a lubricant, and as an analgesic (Grinspoon 1993). The earliest work to find the active ingredient began in the late 19th century after reports from Dr. O'Shaugnessy during his travels in India. In the true spirit of a responsible clinical researcher, before testing on humans, Dr. O'Shaugnessy described the use of hemp on various animals, a practice not standard for physicians at his time. Based on his findings, he believed that certain patients could benefit from the use of cannabis extracts (O'Shaughnessy 1842). His case studies described the use of the drug in humans for rheumatism, hydrophobia, cholera, tetanus, and infantile convulsions. He cautioned, however, of the "delirium occasioned by continued Hemp inebriation," which continues to be a great -but not insurmountable- obstacle for modern pharmacologists synthesizing drugs targeting the endocannabinoid system. He detailed the effects of cannabis preparations for a variety of ailments in a lecture given to the Medical College of Calcutta in 1839. Based on his work, a renewed interest in active cannabis extracts led to scientific inquiry in Europe and the United States, but an active component was not isolated mostly due to lack of effective techniques available at the time. It was not until 1965 that the major psychoactive constituent Δ9-THC by Mechoulam's group (Mechoulam and Gaoni 1965). By the 1970s, many phytocannabinoids were characterized, and it was determined that they were lipid derivatives. Because of the lipophilic nature of these compounds, their mechanism of action was thought to be mediated by their ability to adhere to cellular membranes, much like the proposed mechanism of anesthetic action (Paton 1975). The isolation of Δ9-THC was a key breakthrough in the discovery of an endogenous cannabinoid system because it allowed for the unexpected identification of a highly specific binding site in the body (Devane, Dysarz et al. 1988). This binding site was isolated and cloned in 1990, from both rat and human tissues (Matsuda, Lolait et al. 1990; Gerard, Mollereau et al.

Since it did not seem logical that the body would invest energy in the synthesis of receptors that specifically bind the constituents of this one plant, scientists began looking for compounds produced by the body that could also bind to CB1R. Binding studies with known neurotransmitters and hormones proved to be unfruitful, indicating that a unique ligand was utilizing this newly discovered CB1R. By using a highly specific probe for CB1R labeled with tritium (Devane, Breuer et al. 1992), competitive binding studies in pig brain fractions indicated the presence of endogenous compounds with cannabimimetic activity. Chromatography, nuclear magnetic resonance and mass spectrometry were used to identify arachidonoylethanolamide (Devane, Hanus et al. 1992). An amide group in this newly discovered compound and the historically acknowledged effect of cannabis use, led to the witty alternate name for the very first endocannabinoid 'anandamide', deriving from the Sanskrit word for 'bliss' (Devane, Hanus et al. 1992; Mechoulam 2000). Not only did anandamide work like Δ9-THC in binding assays, it also mimicked its effects on motor

In 1993, shortly after the discovery of anandamide, another cannabinoid receptor was found and cloned from the periphery (rat spleen), and identified mostly on immune cells (Munro, Thomas et al. 1993). It was referred to as the CB2 receptor (CB2R). Two groups, made the separate discovery of another endocannabinoid, 2-arachidonoyl glycerol (2-AG), that was

1991) and was named the cannabinoid 1 receptor (CB1R).

functions, sedation and pain relief (Mechoulam 2000).

capable of binding to both the original CB1R, and to this novel CB2R receptor [(Mechoulam, Ben-Shabat et al. 1995; Sugiura, Kondo et al. 1995) and see **Figure 1** for a timeline].

Fig. 1. **A Historical timeline of the eCB system.** Illustrated above are the major developments leading up to the discovery of an endogenous cannabinoid system, with the pivotal discoveries of the cannabinoid receptors, CB1R and CB2R, and the ligands AEA & 2-AG. Partly summarized from (Mechoulam and Hanus 2000). Receptors made with motifolio.com©.

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 103

Since it is believed that cannabis can be habit forming, evidence suggests that the brain area that processes addictive and reinforcing behaviors, the ventral tegmental area (VTA), contain GABAergic and glutamatergic terminals that express CB1R (Melis, Pistis et al. 2004). A potential, but yet unsubstantiated, role of CB1R in this area may be to facilitate other addictive behaviors such as alcoholism or illicit drug use (Mackie 2005). Cannabis and cannabinoid compounds have also been used as anti-emetics (Darmani 2001). Studies have illustrated that indeed the brain area responsible for emesis, the medullary nuclei of the brainstem (i.e. area postrema) contain high levels of the CB1R predominantly located on axon terminals. It is strongly believed that this anti-emesis may be attributed to CB1R activation in this area (Van Sickle, Oland et al. 2001; Van Sickle, Oland et al. 2003; Martin

In the spinal cord, several studies have been published demonstrating that CB1R is found throughout the gray matter, but at higher densities in the dorsal areas relative to the ventral areas (Herkenham, Lynn et al. 1991; Tsou, Brown et al. 1998; Ong and Mackie 1999; Farquhar-Smith, Egertova et al. 2000; Mackie 2005; Hegyi, Kis et al. 2009). Many of our essential functions depend on an intact and healthy spinal cord, such as sensation (modulated primarily by the dorsal spinal cord) and locomotion (modulated primarily by the ventral spinal cord). This is evident particularly is diseases of the spinal cord or after traumatic injury, in which the most severe cases render the individual incapable of feeling or moving, or even death. At the spinal cord level, endocannabinoid tone and receptor expression appear to play a role in modulating movement (El Manira, Kyriakatos et al. 2008; El Manira and Kyriakatos 2010), but also nociception (Pernia-Andrade, Kato et al. 2009). Therefore, understanding the role of the eCB system in the adult spinal cord is clinically

Though strong evidence exists for neuronal CB1R expression, evidence also exists for its expression on astrocytes in the rat striatum (Rodriguez, Mackie et al. 2001), hippocampus (Navarrete and Araque 2008), and spinal cord (Salio, Doly et al. 2002). In addition, microglia derived from neonatal rat brains, were also found to be immunoreactive for CB1R (Waksman, Olson et al. 1999). RIP-positive or APC-positive oligodendrocytes in healthy adult rat brains and spinal cords, respectively, constitutively express CB1R (Molina-

CB1R is included among the most abundant receptors in the brain, with picomolar ranges per milligram of tissue (as determined from rat brain (Herkenham, Lynn et al. 1991; Pazos, Nunez et al. 2005). Interestingly, compared to the abundance of CB1R, under physiological conditions, the amounts of eCBs (AEA and 2-AG) reach only into the low femtomolar range (Bisogno, Berrendero et al. 1999; Pazos, Nunez et al. 2005). This discrepancy - higher amounts of receptor and lower amounts of endogenous ligands- can be reconciled by understanding the function of the endocannabinoid system as an elegant and efficient negative feedback mechanism to control the levels of neurotransmitters released into the

Neurotransmitters are synthesized in the pre-synaptic neuron, and stored in vesicles ready to be released into the synaptic cleft after depolarization leads to an influx of calcium

relevant, and deserves as much attention as other areas of the CNS.

**4. Role of CB1R activation in adult neurons** 

and Wiley 2004; Mackie 2005).

Holgado, Vela et al. 2002).

synaptic cleft.

By the end of the 20th century, the basic components of the endocannabinoid system- the receptors, endogenous ligands, and the enzymes responsible for their synthesis and degradation- were identified, paving the way for the groundbreaking discoveries that continually emerge, bringing forth the often surprising and unexpected ways in which this system works in the body.

## **3. CB1R localization in the central nervous system**

With the discovery of the endocannabinoid system came the natural question as to what exactly these ligands and receptors are doing in the body. The location and density of CB1R could not only help explain some of the effects of cannabis use, but also has suggested the potential role of the endocannabinoids in learning, memory, motor function, emesis, reward behaviors and pain.

CB1R is a G-protein coupled receptor encoded by a single gene located on chromosome 4 in the mouse, 5 in the rat and 6 in humans**.** The mouse and rat display 95% nucleic acid homology, and 99.5% amino acid homology, while the mouse and human display 90% nucleic acid homology, and 97% amino acid homology (Onaivi, Leonard et al. 2002).

This receptor has been identified in both cortical and subcortical areas, the olfactory bulb, the retina, periaqueductal gray area, the cerebellum and the spinal cord (Mackie 2005). Original autoradiography studies revealed that the substantia nigra contains the highest density of CB1R in the central nervous system (CNS) (Herkenham, Lynn et al. 1991). The substantia nigra is a structure in the midbrain that plays an important role in movement, reward and addiction. CB1R is localized to the GABAergic (GABA = Gamma-aminobutyric acid) axons that project to the substantia nigra from the putamen. CB1R is also found in the caudate putamen, and on axons of medium spiny neurons projecting into the globus pallidus, on excitatory glutamatergic axons projecting from the sub-thalamic nucleus into the substantia nigra (Mailleux, Verslijpe et al. 1992; Sanudo-Pena, Tsou et al. 1997; Mackie 2005).

Much attention has been paid to the hippocampus and CB1R expression mainly because of the striking effects of marijuana on cognitive processes like memory. CB1R is widely distributed in the hippocampal structures. For example, high amounts of the receptor are found in the molecular and granule cell layer of the dentate gyrus (Mackie 2005), in the perisomatic region of CA1 indicative of expression that is post-synpaptic to basket cells, and may also be found on glutamatergic terminals of the perforant path (Kirby, Hampson et al. 1995). In the frontal cortex, double-immunocytochemical labeling experiments revealed GABAergic cholecystokinin (CCK) positive interneurons have somatic immunoreactivity for CB1R (Katona, Sperlagh et al. 1999; Tsou, Mackie et al. 1999). In terms of the laminar distribution within the neocortex high expression is found in layer II, upper III, layer IV and VI. Also it was found that the majority of cells in the neocortex which express the CB1R also express GAD65 (glutamic acid decarboxylase), the enzyme which converts L-glutamate to GABA, thus identifying inhibitory neurons in the CNS. In the cerebellum, there is a very high expression of CB1R in the molecular layer where the Purkinje neuron- parallel fiber synapse is found. Also, electrophysiological experiments infer that there are somatic CB1Rs on basket cells within the cerebellum. Therefore, there is strong evidence to suggest the presence of CB1R on GABAergic and glutamatergic neurons within the cerebellum (Mackie 2005).

By the end of the 20th century, the basic components of the endocannabinoid system- the receptors, endogenous ligands, and the enzymes responsible for their synthesis and degradation- were identified, paving the way for the groundbreaking discoveries that continually emerge, bringing forth the often surprising and unexpected ways in which this

With the discovery of the endocannabinoid system came the natural question as to what exactly these ligands and receptors are doing in the body. The location and density of CB1R could not only help explain some of the effects of cannabis use, but also has suggested the potential role of the endocannabinoids in learning, memory, motor function, emesis, reward

CB1R is a G-protein coupled receptor encoded by a single gene located on chromosome 4 in the mouse, 5 in the rat and 6 in humans**.** The mouse and rat display 95% nucleic acid homology, and 99.5% amino acid homology, while the mouse and human display 90%

This receptor has been identified in both cortical and subcortical areas, the olfactory bulb, the retina, periaqueductal gray area, the cerebellum and the spinal cord (Mackie 2005). Original autoradiography studies revealed that the substantia nigra contains the highest density of CB1R in the central nervous system (CNS) (Herkenham, Lynn et al. 1991). The substantia nigra is a structure in the midbrain that plays an important role in movement, reward and addiction. CB1R is localized to the GABAergic (GABA = Gamma-aminobutyric acid) axons that project to the substantia nigra from the putamen. CB1R is also found in the caudate putamen, and on axons of medium spiny neurons projecting into the globus pallidus, on excitatory glutamatergic axons projecting from the sub-thalamic nucleus into the substantia nigra

Much attention has been paid to the hippocampus and CB1R expression mainly because of the striking effects of marijuana on cognitive processes like memory. CB1R is widely distributed in the hippocampal structures. For example, high amounts of the receptor are found in the molecular and granule cell layer of the dentate gyrus (Mackie 2005), in the perisomatic region of CA1 indicative of expression that is post-synpaptic to basket cells, and may also be found on glutamatergic terminals of the perforant path (Kirby, Hampson et al. 1995). In the frontal cortex, double-immunocytochemical labeling experiments revealed GABAergic cholecystokinin (CCK) positive interneurons have somatic immunoreactivity for CB1R (Katona, Sperlagh et al. 1999; Tsou, Mackie et al. 1999). In terms of the laminar distribution within the neocortex high expression is found in layer II, upper III, layer IV and VI. Also it was found that the majority of cells in the neocortex which express the CB1R also express GAD65 (glutamic acid decarboxylase), the enzyme which converts L-glutamate to GABA, thus identifying inhibitory neurons in the CNS. In the cerebellum, there is a very high expression of CB1R in the molecular layer where the Purkinje neuron- parallel fiber synapse is found. Also, electrophysiological experiments infer that there are somatic CB1Rs on basket cells within the cerebellum. Therefore, there is strong evidence to suggest the presence of CB1R on GABAergic

nucleic acid homology, and 97% amino acid homology (Onaivi, Leonard et al. 2002).

(Mailleux, Verslijpe et al. 1992; Sanudo-Pena, Tsou et al. 1997; Mackie 2005).

and glutamatergic neurons within the cerebellum (Mackie 2005).

system works in the body.

behaviors and pain.

**3. CB1R localization in the central nervous system** 

Since it is believed that cannabis can be habit forming, evidence suggests that the brain area that processes addictive and reinforcing behaviors, the ventral tegmental area (VTA), contain GABAergic and glutamatergic terminals that express CB1R (Melis, Pistis et al. 2004). A potential, but yet unsubstantiated, role of CB1R in this area may be to facilitate other addictive behaviors such as alcoholism or illicit drug use (Mackie 2005). Cannabis and cannabinoid compounds have also been used as anti-emetics (Darmani 2001). Studies have illustrated that indeed the brain area responsible for emesis, the medullary nuclei of the brainstem (i.e. area postrema) contain high levels of the CB1R predominantly located on axon terminals. It is strongly believed that this anti-emesis may be attributed to CB1R activation in this area (Van Sickle, Oland et al. 2001; Van Sickle, Oland et al. 2003; Martin and Wiley 2004; Mackie 2005).

In the spinal cord, several studies have been published demonstrating that CB1R is found throughout the gray matter, but at higher densities in the dorsal areas relative to the ventral areas (Herkenham, Lynn et al. 1991; Tsou, Brown et al. 1998; Ong and Mackie 1999; Farquhar-Smith, Egertova et al. 2000; Mackie 2005; Hegyi, Kis et al. 2009). Many of our essential functions depend on an intact and healthy spinal cord, such as sensation (modulated primarily by the dorsal spinal cord) and locomotion (modulated primarily by the ventral spinal cord). This is evident particularly is diseases of the spinal cord or after traumatic injury, in which the most severe cases render the individual incapable of feeling or moving, or even death. At the spinal cord level, endocannabinoid tone and receptor expression appear to play a role in modulating movement (El Manira, Kyriakatos et al. 2008; El Manira and Kyriakatos 2010), but also nociception (Pernia-Andrade, Kato et al. 2009). Therefore, understanding the role of the eCB system in the adult spinal cord is clinically relevant, and deserves as much attention as other areas of the CNS.

Though strong evidence exists for neuronal CB1R expression, evidence also exists for its expression on astrocytes in the rat striatum (Rodriguez, Mackie et al. 2001), hippocampus (Navarrete and Araque 2008), and spinal cord (Salio, Doly et al. 2002). In addition, microglia derived from neonatal rat brains, were also found to be immunoreactive for CB1R (Waksman, Olson et al. 1999). RIP-positive or APC-positive oligodendrocytes in healthy adult rat brains and spinal cords, respectively, constitutively express CB1R (Molina-Holgado, Vela et al. 2002).

#### **4. Role of CB1R activation in adult neurons**

CB1R is included among the most abundant receptors in the brain, with picomolar ranges per milligram of tissue (as determined from rat brain (Herkenham, Lynn et al. 1991; Pazos, Nunez et al. 2005). Interestingly, compared to the abundance of CB1R, under physiological conditions, the amounts of eCBs (AEA and 2-AG) reach only into the low femtomolar range (Bisogno, Berrendero et al. 1999; Pazos, Nunez et al. 2005). This discrepancy - higher amounts of receptor and lower amounts of endogenous ligands- can be reconciled by understanding the function of the endocannabinoid system as an elegant and efficient negative feedback mechanism to control the levels of neurotransmitters released into the synaptic cleft.

Neurotransmitters are synthesized in the pre-synaptic neuron, and stored in vesicles ready to be released into the synaptic cleft after depolarization leads to an influx of calcium

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 105

**A)** 

**B)** 

Fig. 3. **Endocannabinoids act as retrograde messengers in the CNS. (A)** Neurotransmitters bind to their postsynaptic receptors causing the synthesis of AEA or 2-AG via their synthetic enzymes, NAT/NAPE-PLD and PLC DAGL, respectively, before traveling retrogradely to bind to CB1Rs. **(B)** Inhibition of voltage-dependent calcium channels is one way by which neurotransmitter release probability is decreased. Binding of ligand to CB1R can result in the inactivation of N and P/Q- type, but not L-type calcium (Ca2+) channels (Caulfield and

Brown 1992; Mackie and Hille 1992; Mackie, Devane et al. 1993; Pertwee 1997). The

particular channel involved is related to the brain region: in rat striatum, CB1Rs modulate N-type Ca2+ channels (Huang, Lo et al. 2001; Schlicker and Kathmann 2001) and not L, P or Q-type Ca2+ channels. In cultured rat hippocampal neurons, the CB1R modulates N- and Q-, but not P-type calcium channels (Sullivan 1999; Schlicker and Kathmann 2001). However, CB1R does not modulate any of the voltage- dependent Ca2+ channels found in the nucleus accumbens (Robbe, Alonso et al. 2001; Schlicker and Kathmann 2001). In contrast, newer evidence suggests that CB1R activation modulates all of the voltage-dependent Ca2+

channels found at the granule cell-Purkinje cell synapse of the cerebellum: the N-, P/Q- and

R-type Ca2+ channels (Brown, Safo et al. 2004).

through voltage-dependent calcium channels. In contrast, eCBs are synthesized on demand in the post-synaptic neuron using lipid precursors from cell membranes (Di Marzo, Bifulco et al. 2004) - **Figure 2**.

Fig. 2. The enzymes responsible for the synthesis and degradation of the two major eCBs, AEA and 2-AG. They are made on-demand from membrane lipid precursors in the post-synaptic neuron. The endocannabinoid membrane transporter (EMT) facilitates their re-uptake into either the post-synaptic (2-AG) or pre-synaptic (AEA) neuron for degradation by MAGL, or FAAH, respectively (Di Marzo, Bifulco et al. 2004; El Manira and Kyriakatos 2010)

Endocannabinoids readily pass through the post-synaptic membrane, travel retrogradely into the synaptic cleft, and bind to pre-synaptically located CB1Rs (Wilson and Nicoll 2002). As a G-protein coupled receptor, activation of CB1R by the endocannabinoids results in various cellular consequences, two of which are the ability to inhibit voltage-dependent calcium channels, or activate inwardly rectifying potassium channels. These processes affect the pre-synaptic neuron by ultimately decreasing the probability of neurotransmitter release (**Figure 3**).

The magnitude and duration of CB1R activation affects the machinery responsible for the release of several neurotransmitters such as glutamate, GABA, glycine, acetylcholine, noradrenaline and serotonin (Szabo and Schlicker 2005). Therefore, within a neuronal circuit, cells are able to regulate the strength of their synaptic inputs by on-demand release of eCBs which can then bind to CB1R (Freund, Katona et al. 2003). The high abundance of CB1Rs coupled with the relatively low-levels of detectable eCBs can be attributed to the fact that released ligand does not accumulate, but rather acts rapidly and transiently to mediate synaptic plasticity (Pazos, Nunez et al. 2005). In order to achieve such a highly efficient modulation of activity without accumulation of ligand, there must be a high density of receptors. This is precisely the state of the endocannabinoid system under physiological conditions.

through voltage-dependent calcium channels. In contrast, eCBs are synthesized on demand in the post-synaptic neuron using lipid precursors from cell membranes (Di Marzo, Bifulco

Fig. 2. The enzymes responsible for the synthesis and degradation of the two major eCBs, AEA and 2-AG. They are made on-demand from membrane lipid precursors in the post-synaptic neuron. The endocannabinoid membrane transporter (EMT) facilitates their re-uptake into either the post-synaptic (2-AG) or pre-synaptic (AEA) neuron for degradation by MAGL, or

Endocannabinoids readily pass through the post-synaptic membrane, travel retrogradely into the synaptic cleft, and bind to pre-synaptically located CB1Rs (Wilson and Nicoll 2002). As a G-protein coupled receptor, activation of CB1R by the endocannabinoids results in various cellular consequences, two of which are the ability to inhibit voltage-dependent calcium channels, or activate inwardly rectifying potassium channels. These processes affect the pre-synaptic neuron by ultimately decreasing the probability of neurotransmitter release

The magnitude and duration of CB1R activation affects the machinery responsible for the release of several neurotransmitters such as glutamate, GABA, glycine, acetylcholine, noradrenaline and serotonin (Szabo and Schlicker 2005). Therefore, within a neuronal circuit, cells are able to regulate the strength of their synaptic inputs by on-demand release of eCBs which can then bind to CB1R (Freund, Katona et al. 2003). The high abundance of CB1Rs coupled with the relatively low-levels of detectable eCBs can be attributed to the fact that released ligand does not accumulate, but rather acts rapidly and transiently to mediate synaptic plasticity (Pazos, Nunez et al. 2005). In order to achieve such a highly efficient modulation of activity without accumulation of ligand, there must be a high density of receptors. This is precisely the state of the endocannabinoid system under physiological

FAAH, respectively (Di Marzo, Bifulco et al. 2004; El Manira and Kyriakatos 2010)

et al. 2004) - **Figure 2**.

(**Figure 3**).

conditions.

Fig. 3. **Endocannabinoids act as retrograde messengers in the CNS. (A)** Neurotransmitters bind to their postsynaptic receptors causing the synthesis of AEA or 2-AG via their synthetic enzymes, NAT/NAPE-PLD and PLC DAGL, respectively, before traveling retrogradely to bind to CB1Rs. **(B)** Inhibition of voltage-dependent calcium channels is one way by which neurotransmitter release probability is decreased. Binding of ligand to CB1R can result in the inactivation of N and P/Q- type, but not L-type calcium (Ca2+) channels (Caulfield and Brown 1992; Mackie and Hille 1992; Mackie, Devane et al. 1993; Pertwee 1997). The particular channel involved is related to the brain region: in rat striatum, CB1Rs modulate N-type Ca2+ channels (Huang, Lo et al. 2001; Schlicker and Kathmann 2001) and not L, P or Q-type Ca2+ channels. In cultured rat hippocampal neurons, the CB1R modulates N- and Q-, but not P-type calcium channels (Sullivan 1999; Schlicker and Kathmann 2001). However, CB1R does not modulate any of the voltage- dependent Ca2+ channels found in the nucleus accumbens (Robbe, Alonso et al. 2001; Schlicker and Kathmann 2001). In contrast, newer evidence suggests that CB1R activation modulates all of the voltage-dependent Ca2+ channels found at the granule cell-Purkinje cell synapse of the cerebellum: the N-, P/Q- and R-type Ca2+ channels (Brown, Safo et al. 2004).

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 107

downregulated in the amygdala and Periaqueductal Gray Area (Garcia-Ovejero, Arevalo-Martin et al. 2009; Knerlich-Lukoschus, Noack et al. 2011). In healthy spinal cords, several studies indicate that there are very low levels of CB2R, but peripheral nerve injury, for example, leads to significant upregulation of this receptor, corresponding to significant microglial activation in the spinal cord (Zhang, Hoffert et al. 2003; Romero-Sandoval, Nutile-McMenemy et al. 2008). Microglial cells contribute to the inflammatory response by producing and secreting the pro-inflammatory cytokines that contribute to excitotoxic damage in the CNS, but also to the differentiation of pathogenic lymphocytes entering the CNS (Arevalo-Martin, Garcia-Ovejero et al. 2008). Activation of CB2R in cultured microglial cells inhibits these inflammatory cytokines, making CB2R activation an anti-inflammatory target. However, the potential role of CB2R in microglial cells following injury is not clear. Cultured rat microglial cells can produce the eCBs 2-AG and AEA, which in turn auto-

Whether these changes reflect an adaptive defense mechanism or contribute to pathology is still a matter of debate. These studies implicate CB1R and CB2R as double edged swords for CNS insult, and whether their activation promotes protection or contributes to damage likely depends on the etiology and progression of the disease or injury, but also in the

Progenitor cells in the adult CNS are promising targets as endogenous repair mechanisms following insult, and their proliferation and differentiation may provide an avenue to do so. The functional significance of constitutive or pathologically-induced neurogenesis in the adult brain has been associated with wide ranging processes such as memory formation and consolidation, depression, anxiety, and seizure- like activity (Ming and Song 2011). Endocannabinoid system elements have recently been discovered in adult brain progenitor cells (Aguado, Monory et al. 2005; Aguado, Palazuelos et al. 2006; Palazuelos, Aguado et al. 2006). There is an emerging and critical role for the eCB system and specifically, CB1R in adult brain progenitor cells, revealing a novel strategy to help the brain repair itself (Galve-

In the adult brain, the subgranular zone (SGZ) of the hippocampus, and the subventricular zone (SVZ) contain two different populations of progenitor cells. The first population is referred to as the type 1 or type B cells (SGZ and SVZ, respectively). These cells resemble their developmental counterparts; the radial glia. They are characterized by their slow proliferation kinetics, their morphological hallmarks (tiny processes extending from their somata in the SVZ), and these cells express both Nestin and Glial Fibrillary Acidic Protein (GFAP). The type 2 or C cells (SGZ and SVZ, respectively) are actively dividing, non-radial cells that maintain their Nestin expression, but do not express GFAP. They are occasionally positive for the immature neuronal marker Doublecortin (DCX). Ablation studies indicate that these two different populations are distinct in their characteristics, but they are developmentally connected to one another. The type 1, B cells give rise to the type 2, C cells, and if the latter are destroyed, they can eventually be replenished by the former (Suh, Deng et al. 2009). These progenitor cells give birth to new neurons continually throughout

stimulate their CB2Rs to induce proliferation (Carrier, Kearn et al. 2004).

localization of each receptor on specific cell types.

**6. Adult CNS progenitor cells and CB1R** 

adulthood, in a process known as adult neurogenesis.

Roperh, Aguado et al. 2007).

Furthermore, CB1R activation causes cAMP levels to drop because CB1R is negatively coupled to adenylate cyclase (AC) through heterotrimeric Gi/o proteins, (Matsuda, Lolait et al. 1990; Munro, Thomas et al. 1993; Guzman, Sanchez et al. 2002). CB1R activation is also associated with activation of extracellular signal-related kinase (ERK) (Bouaboula, Poinot-Chazel et al. 1995; Wartmann, Campbell et al. 1995) c-Jun N-terminal kinase (Jnk) p38 mitogen activated-protein kinase (p38) (Rueda, Navarro et al. 2002), protein kinase B (Gomez del Pulgar, Velasco et al. 2000), and increased levels of the second messenger ceramide (Sanchez, Galve-Roperh et al. 1998; Guzman, Sanchez et al. 2002) (**Figure 4**). These pathways have been shown to modulate various cellular functions including cell fate, apoptosis and survival in different cell types (Guzman, Sanchez et al. 2001).

Fig. 4. **The effects of pre-synaptic CB1 receptor activation**. CB1R activation on pre-synaptic neurons inhibits voltage dependent calcium channels, and adenylate cyclase, but can also activate inwardly rectifying potassium channels, and the MAPK pathway. (Image adapted from DiMarzo et al. 2004, and Guzman et al. 2002. and created with motifolio.com©)
