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

106 Pharmacology

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,

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©)

Because of the ubiquitous expression of the receptor throughout the CNS, several preclinical and clinical studies have addressed the potential therapeutic value in modulating the endocannabinoid system for analgesia, weight loss, appetite stimulation, neuroprotection after ischemic injuries, and for anti-emetic, anti-epileptic and antispasmodic purposes (Nogueiras, Diaz-Arteaga et al. 2009; Bisogno and Di Marzo 2010; Karst, Wippermann et al. 2010; Scotter, Abood et al. 2010). The premise of many of these therapeutic approaches lies in the neuromodulatory function of CB1R, or in the anti-

During disease or following injury, cannabinoid receptor expression and levels of eCBs are altered. For example, after rat spinal cord injury, cannabinoid receptor expression is altered at the spinal level, but also in brain areas: in the spinal cord, CB1R becomes expressed in reactive astrocytes, and CB2R becomes strongly upregulated in microglia, astrocytes and macrophages. In the brain, CB1R is upregulated in thalamic and hippocampal areas, while

**5. Role of cannabinoid receptors during pathological states** 

inflammatory effects on CB2R activation.

apoptosis and survival in different cell types (Guzman, Sanchez et al. 2001).

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-Roperh, Aguado et al. 2007).

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 adulthood, in a process known as adult neurogenesis.

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 109

antagonist AM251 to wild-type mice promoted proliferation of type 2b/3 DCX(+) cells 7

Fig. 5. CB1R is expressed throughout neuronal development (Harkany, Guzman et al. 2007; Harkany, Keimpema et al. 2008), and also at all stages of adult hippocampal neurogenesis. It is not clear whether CB1R is enriched in certain progenitor populations, and if so, how endogenous cannabinoids differentially affect these populations. Equally compelling is how exogenous CB1R agonists or antagonists may affect these different populations, and what the functional outcomes of such interventions may be. Image

Clarification through additional studies must be made to reconcile these seemingly disparate results. Species, sex and strain of the animals used, chronic versus acute treatment with cannabinergic drugs, specificity, dose/concentration of cannabinergic drugs, BrdU injection protocol and immunohistochemical markers must all be considered when

days after BrdU administration (**Figure 5**).

created with motifolio.com©.

It is imperative to distinguish these progenitor populations when assessing the role of the various eCB components in the neurogenic process. This distinction is rarely made in the literature, and yet it is very plausible that the various eCB system components affect these progenitor populations differently. The results from the following studies indicate that the distinct processes involved in adult brain neurogenesis cannot be grouped together with regards to endocannabinoid modulation.

The role of the eCB system, and in particular CB1R, on adult brain neurogenesis is not clear, partly because the separation between effects on progenitor proliferation and neuronal differentiation have not always been made. A study published in 2004 concluded that that there is defective neurogenesis in the CB1R knockout (KO) mouse (Jin, Xie et al. 2004). A major limitation to this study is that the authors equated changes in BrdU (thymidine analog) incorporation with changes in neurogenesis. Their data strongly support the view that CB1R is critical in progenitor proliferation in the hippocampus, but nothing more can be deduced with regards to which progenitor population is affected, nor about neuronal differentiation and maturation of the remaining progenitor cells.

Pharmacological studies in wild-type mice support the conclusions from CB1R KO mice. Treatment with CB1R agonists (either with the endocannabinoid anandamide, the synthetic agonists WIN 55, 212-2 or HU-210) increased the number of BrdU positive(+)/NeuN negative(-) hippocampal cells, but decreased the number of co-labeled, newly generated BrdU(+)/NeuN(+) neurons *in vivo* (Rueda, Navarro et al. 2002; Aguado, Palazuelos et al. 2006; Galve-Roperh, Aguado et al. 2006). Furthermore, these studies showed that a CB1R antagonist, SR141716, reversed the agonist actions- the number of colabeled cells increased, while BrdU(+)/NeuN(-) cells decreased. Similarly, in a study by Jiang et al, 2005, CB1R activation resulted in increased BrdU(+) cells, which was interpreted as enhanced neurogenesis by cannabinoids; however, the authors themselves never show increases in co-labeled cells, and also point out that relative to no treatment, CB1R agonists do not change the percentage of cells expressing immature neuronal markers (Jiang, Zhang et al. 2005).

Adult hippocampal progenitor cells from mouse brains express CB1R *in vitro* and *in vivo* (Aguado, Monory et al. 2005; Aguado, Palazuelos et al. 2006). CB1R activation induced proliferation of these progenitors assessed by quantifying the amount of cells expressing Nestin and incorporating the thymidine analog BrdU. Interestingly, these studies showed CB1R and FAAH are selectively enriched in type 1 (Nestin(+)/GFAP(+)) progenitors *in vivo* compared to type 2 (Nestin(+)/ GFAP(-)) (Aguado, Palazuelos et al. 2006). Utilizing various markers for immature neurons and glia, CB1R activation appears to promote astroglial differentiation, while inhibition of the receptor appears to promote neuronal differentiation (Aguado, Palazuelos et al. 2006). In contrast, a recent study indicated that CB1R was preferentially expressed on type 2b/3 cells that are also expressing DCX, suggesting that CB1Rs have a role in later stages of neuronal differentiation, and migration of the nascent neuron (Wolf, Bick-Sander et al. 2010). This study examined the levels of DCX expressing cells in the hippocampi of CB1R KO mice, and also in Nestin-GFP reporter mice treated with the CB1R antagonist AM251. According to the authors, genetic deletion of CB1R resulted in increased proliferation but decreased net neurogenesis relative to wild-type mice. But, administration of the CB1R specific

It is imperative to distinguish these progenitor populations when assessing the role of the various eCB components in the neurogenic process. This distinction is rarely made in the literature, and yet it is very plausible that the various eCB system components affect these progenitor populations differently. The results from the following studies indicate that the distinct processes involved in adult brain neurogenesis cannot be grouped together with

The role of the eCB system, and in particular CB1R, on adult brain neurogenesis is not clear, partly because the separation between effects on progenitor proliferation and neuronal differentiation have not always been made. A study published in 2004 concluded that that there is defective neurogenesis in the CB1R knockout (KO) mouse (Jin, Xie et al. 2004). A major limitation to this study is that the authors equated changes in BrdU (thymidine analog) incorporation with changes in neurogenesis. Their data strongly support the view that CB1R is critical in progenitor proliferation in the hippocampus, but nothing more can be deduced with regards to which progenitor population is affected, nor about neuronal

Pharmacological studies in wild-type mice support the conclusions from CB1R KO mice. Treatment with CB1R agonists (either with the endocannabinoid anandamide, the synthetic agonists WIN 55, 212-2 or HU-210) increased the number of BrdU positive(+)/NeuN negative(-) hippocampal cells, but decreased the number of co-labeled, newly generated BrdU(+)/NeuN(+) neurons *in vivo* (Rueda, Navarro et al. 2002; Aguado, Palazuelos et al. 2006; Galve-Roperh, Aguado et al. 2006). Furthermore, these studies showed that a CB1R antagonist, SR141716, reversed the agonist actions- the number of colabeled cells increased, while BrdU(+)/NeuN(-) cells decreased. Similarly, in a study by Jiang et al, 2005, CB1R activation resulted in increased BrdU(+) cells, which was interpreted as enhanced neurogenesis by cannabinoids; however, the authors themselves never show increases in co-labeled cells, and also point out that relative to no treatment, CB1R agonists do not change the percentage of cells expressing immature neuronal

Adult hippocampal progenitor cells from mouse brains express CB1R *in vitro* and *in vivo* (Aguado, Monory et al. 2005; Aguado, Palazuelos et al. 2006). CB1R activation induced proliferation of these progenitors assessed by quantifying the amount of cells expressing Nestin and incorporating the thymidine analog BrdU. Interestingly, these studies showed CB1R and FAAH are selectively enriched in type 1 (Nestin(+)/GFAP(+)) progenitors *in vivo* compared to type 2 (Nestin(+)/ GFAP(-)) (Aguado, Palazuelos et al. 2006). Utilizing various markers for immature neurons and glia, CB1R activation appears to promote astroglial differentiation, while inhibition of the receptor appears to promote neuronal differentiation (Aguado, Palazuelos et al. 2006). In contrast, a recent study indicated that CB1R was preferentially expressed on type 2b/3 cells that are also expressing DCX, suggesting that CB1Rs have a role in later stages of neuronal differentiation, and migration of the nascent neuron (Wolf, Bick-Sander et al. 2010). This study examined the levels of DCX expressing cells in the hippocampi of CB1R KO mice, and also in Nestin-GFP reporter mice treated with the CB1R antagonist AM251. According to the authors, genetic deletion of CB1R resulted in increased proliferation but decreased net neurogenesis relative to wild-type mice. But, administration of the CB1R specific

regards to endocannabinoid modulation.

markers (Jiang, Zhang et al. 2005).

differentiation and maturation of the remaining progenitor cells.

antagonist AM251 to wild-type mice promoted proliferation of type 2b/3 DCX(+) cells 7 days after BrdU administration (**Figure 5**).

Fig. 5. CB1R is expressed throughout neuronal development (Harkany, Guzman et al. 2007; Harkany, Keimpema et al. 2008), and also at all stages of adult hippocampal neurogenesis. It is not clear whether CB1R is enriched in certain progenitor populations, and if so, how endogenous cannabinoids differentially affect these populations. Equally compelling is how exogenous CB1R agonists or antagonists may affect these different populations, and what the functional outcomes of such interventions may be. Image created with motifolio.com©.

Clarification through additional studies must be made to reconcile these seemingly disparate results. Species, sex and strain of the animals used, chronic versus acute treatment with cannabinergic drugs, specificity, dose/concentration of cannabinergic drugs, BrdU injection protocol and immunohistochemical markers must all be considered when

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 111

not. There are several clinical examples where new neuron formation in the adult spinal cord could potentially ameliorate disease symptoms or progression, or replace damaged neurons following trauma. Replacement of dead or damaged neurons in the compromised spinal cord may be able to promote functional motor recovery, but also reduce pain (Hofstetter, Holmstrom et al. 2005; Scholz, Broom et al. 2005; Ohori, Yamamoto et al. 2006; Meisner, Marsh et al. 2010). Manipulating the spinal cord environment to coerce neurogenesis from endogenous progenitors is a promising therapeutic intervention, which may bypass the many obstacles inherent to transplantation of exogenous stem/progenitor

Several models propose distinct locations for the endogenous spinal cord progenitors, and how they respond to physiological and pathological stimuli (Namiki and Tator 1999; Horner, Power et al. 2000; Horky, Galimi et al. 2006; Meletis, Barnabe-Heider et al. 2008; Hamilton, Truong et al. 2009; Barnabe-Heider, Goritz et al. 2010; Hugnot and Franzen 2011). The overwhelming majority of progenitor cells do not differentiate into neurons *in vivo*. Nevertheless, these progenitors have neurogenic potential revealed from *in vitro* studies, but also from *in vivo* transplantation studies. Progenitors isolated from all levels and areas of the adult spinal cord can give rise to neurons in culture (Weiss, Dunne et al. 1996; Yamamoto, Yamamoto et al. 2001). When spinal cord progenitors were transplanted into the hippocampus- a pro-neurogenic environment, they readily formed neurons (Shihabuddin, Horner et al. 2000). These studies imply that the spinal cord environment is restricting the neurogenic potential of the endogenous progenitors, and astrocytes may be one of the

New evidence is emerging to challenge the idea that new neurons cannot be generated in the adult spinal cord. Direct injury to the spinal cord results in massive progenitor proliferation leading to astrocyte differentiation, and a massive inflammatory response which contributes to glial scar formation (Barnabe-Heider, Goritz et al. 2010; Wang, Cheng et al. 2011). This injured environment has been demonstrated as non-neurogenic (Yamamoto, Nagao et al. 2001; Hannila, Siddiq et al. 2007); however, there are instances in which an environment filled with inflammatory cytokines can still elicit neurogenesis in the adult spinal cord. For example, in an experimental rat model of multiple sclerosis (experimental autoimmune encephalomyelitis), newly generated neurons migrated towards the neuroinflammatory lesion (Danilov, Covacu et al. 2006). Also there are instances of indirect injury to the adult spinal cord, such as dorsal rhizotomy (cutting of the dorsal root at the cervical spinal level) in which neurogenesis is observed in the dorsal horn at the corresponding spinal level (Vessal, Aycock et al. 2007). Recent papers showed that in non-injured, intact adult spinal cords, immature neurons can be found in the area surrounding the central canal (Shechter, Ziv et al. 2007; Marichal, Garcia et al. 2009), but also throughout the spinal cord, with a preferential dorsal gray matter localization and exclusive GABAergic phenotype (Shechter, Ziv et al. 2007; Shechter, Baruch et al. 2011). The exact roles of these immature neurons in the healthy spinal cord have not been determined, but may indicate physiological roles for new GABAergic neurons in nociception (Shechter, Baruch et al. 2011), and also for movement. The existence of these cells is exciting, as it sets the tone for more intensive studies to characterize their function

cells (Obermair, Schroter et al. 2008).

culprits (Song, Stevens et al. 2002).

and promote their differentiation.

interpreting the many studies published on CB1R's role on adult hippocampal neurogenesis. Table 1 summarizes several knock-out mice that have been developed that target endocannabinoid system components, and the consequences on progenitor proliferation, neuronal differentiation and glial differentiation.


Table 1. **eCB Knock-out mice and adult CNS progenitor cells.** a (Aguado, Palazuelos et al. 2006); b(Jin, Xie et al. 2004); c(Palazuelos, Aguado et al. 2006); d(Gao, Vasilyev et al. 2010); KO= Knockout; SVZ = subventricular zone. The apparent conflicting results in the adult CB1 -/- brains may be attributed to the interpretation of 'neurogenesis'( see Section 6).

## **7. Neurogenesis in the adult spinal cord**

Compared to the brain, even though progenitor cells also exist in the adult spinal cord, the spinal cord environment does not seem to support robust constitutive neurogenesis, nor does it seem to support neurogenesis following region specific injury or disease. Though injury results in different functional consequences for the brain and spinal cord, it is not clear why one region of the CNS is capable of generating new neurons, while another area is

interpreting the many studies published on CB1R's role on adult hippocampal neurogenesis. Table 1 summarizes several knock-out mice that have been developed that target endocannabinoid system components, and the consequences on progenitor

Table 1. **eCB Knock-out mice and adult CNS progenitor cells.** a (Aguado, Palazuelos et al. 2006); b(Jin, Xie et al. 2004); c(Palazuelos, Aguado et al. 2006); d(Gao, Vasilyev et al. 2010); KO= Knockout; SVZ = subventricular zone. The apparent conflicting results in the adult CB1 -/- brains may be attributed to the interpretation of 'neurogenesis'( see Section 6).

Compared to the brain, even though progenitor cells also exist in the adult spinal cord, the spinal cord environment does not seem to support robust constitutive neurogenesis, nor does it seem to support neurogenesis following region specific injury or disease. Though injury results in different functional consequences for the brain and spinal cord, it is not clear why one region of the CNS is capable of generating new neurons, while another area is

**7. Neurogenesis in the adult spinal cord** 

proliferation, neuronal differentiation and glial differentiation.

not. There are several clinical examples where new neuron formation in the adult spinal cord could potentially ameliorate disease symptoms or progression, or replace damaged neurons following trauma. Replacement of dead or damaged neurons in the compromised spinal cord may be able to promote functional motor recovery, but also reduce pain (Hofstetter, Holmstrom et al. 2005; Scholz, Broom et al. 2005; Ohori, Yamamoto et al. 2006; Meisner, Marsh et al. 2010). Manipulating the spinal cord environment to coerce neurogenesis from endogenous progenitors is a promising therapeutic intervention, which may bypass the many obstacles inherent to transplantation of exogenous stem/progenitor cells (Obermair, Schroter et al. 2008).

Several models propose distinct locations for the endogenous spinal cord progenitors, and how they respond to physiological and pathological stimuli (Namiki and Tator 1999; Horner, Power et al. 2000; Horky, Galimi et al. 2006; Meletis, Barnabe-Heider et al. 2008; Hamilton, Truong et al. 2009; Barnabe-Heider, Goritz et al. 2010; Hugnot and Franzen 2011). The overwhelming majority of progenitor cells do not differentiate into neurons *in vivo*. Nevertheless, these progenitors have neurogenic potential revealed from *in vitro* studies, but also from *in vivo* transplantation studies. Progenitors isolated from all levels and areas of the adult spinal cord can give rise to neurons in culture (Weiss, Dunne et al. 1996; Yamamoto, Yamamoto et al. 2001). When spinal cord progenitors were transplanted into the hippocampus- a pro-neurogenic environment, they readily formed neurons (Shihabuddin, Horner et al. 2000). These studies imply that the spinal cord environment is restricting the neurogenic potential of the endogenous progenitors, and astrocytes may be one of the culprits (Song, Stevens et al. 2002).

New evidence is emerging to challenge the idea that new neurons cannot be generated in the adult spinal cord. Direct injury to the spinal cord results in massive progenitor proliferation leading to astrocyte differentiation, and a massive inflammatory response which contributes to glial scar formation (Barnabe-Heider, Goritz et al. 2010; Wang, Cheng et al. 2011). This injured environment has been demonstrated as non-neurogenic (Yamamoto, Nagao et al. 2001; Hannila, Siddiq et al. 2007); however, there are instances in which an environment filled with inflammatory cytokines can still elicit neurogenesis in the adult spinal cord. For example, in an experimental rat model of multiple sclerosis (experimental autoimmune encephalomyelitis), newly generated neurons migrated towards the neuroinflammatory lesion (Danilov, Covacu et al. 2006). Also there are instances of indirect injury to the adult spinal cord, such as dorsal rhizotomy (cutting of the dorsal root at the cervical spinal level) in which neurogenesis is observed in the dorsal horn at the corresponding spinal level (Vessal, Aycock et al. 2007). Recent papers showed that in non-injured, intact adult spinal cords, immature neurons can be found in the area surrounding the central canal (Shechter, Ziv et al. 2007; Marichal, Garcia et al. 2009), but also throughout the spinal cord, with a preferential dorsal gray matter localization and exclusive GABAergic phenotype (Shechter, Ziv et al. 2007; Shechter, Baruch et al. 2011). The exact roles of these immature neurons in the healthy spinal cord have not been determined, but may indicate physiological roles for new GABAergic neurons in nociception (Shechter, Baruch et al. 2011), and also for movement. The existence of these cells is exciting, as it sets the tone for more intensive studies to characterize their function and promote their differentiation.

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 113

**Hoechst CB1R**

**Hoechst CB1R Nestin**

Fig. 7. Primary adult spinal cord cultures from rats contain Nestin(+) progenitor cells (red), which also express CB1R (green). The role of CB1R on these progenitors has not been examined, and further studies are needed to determine how the receptor is involved in progenitor cell quiescence, proliferation or differentiation. Image obtained after 6 days *in* 

In response to injury, not only do progenitor cells proliferate in the spinal cord (Frisen, Johansson et al. 1995; Johansson, Momma et al. 1999; Namiki and Tator 1999; Shibuya, Miyamoto et al. 2002), but levels of endocannabinoids, receptors and enzymes are also altered as described earlier (in Section 4). Rigorous studies are needed to address if and how adult spinal cord progenitor cells respond to endogenous cannabinoid tone or to exogenously administered cannabinoids. Does endocannabinoid tone contribute to the nonneurogenic spinal cord environment? Are endo/exo-cannabinoids capable of promoting spinal cord neurogenesis or gliogenesis? These are just a few critical and novel avenues for

**9. The effect of chronic cannabinergic drug use on the CNS- implications for** 

Cannabis is used both acutely and chronically for recreational or medicinal purposes. There is controversy regarding medical marijuana because of the documented cognitive side effects of chronic recreational use (Jager and Ramsey 2008; Hester, Nestor et al. 2009; Battisti, Roodenrys et al. 2010). However, all drugs come with a risk-benefit consideration, and a plethora of historical and emerging evidence indicates that the medicinal value of cannabis cannot be ignored. Many studies have demonstrated that endocannabinoids and application of exogenous cannabinoids (usually mixed CB1R/CB2R agonists) reduce pain sensation (Guindon and Hohmann 2009). Presently, such an approach is becoming more clinically accepted for treating chronic pain states (Aggarwal, Carter et al. 2009; Karst, Wippermann et al. 2010; Lynch and Campbell 2011). While CB2R activation attenuates nociception mostly by modulating the inflammatory response (Guindon and Hohmann 2008), the role of CB1R is more complex because its location on various cells along the pain pathways appears to contribute differently to nociception. Moreover, many cannabinergic drugs are not only mixed agonists, but may bind non-specifically to other receptors, including TRP-channels

The use of several CB1R knock-outs (global and conditional) has helped to clarify the role of these receptors in nociception. Recent worked demonstrated that cannabinoids mediate analgesia by activating CB1Rs located on peripheral nociceptors (dorsal root ganglia sensory neurons) (Agarwal, Pacher et al. 2007). Interestingly, by using *in vitro* spinal cord slices and *in vivo* recordings of dorsal horn neurons, activation of CB1Rs on spinal cord dorsal horn

potentially promoting neurogenesis in the adult spinal cord.

**Hoechst Nestin**

(Patwardhan, Jeske et al. 2006; Patil, Patwardhan et al. 2011).

*vitro* with 63X objective.

**the treatment of chronic pain** 

Fig. 6. Transverse section of an adult mouse spinal cord, depicting a model for progenitor cell and immature neuron location. Based on the work by Shechter et al, 2007, 2011, the majority of the GABAergic, BrdU(+)/ DCX(+) immature neurons reside in the gray matter of the dorsal horn. Under physiological conditions, the levels of these cells depend on the type of and exposure to sensory environmental enrichment. Image generated with motifolio.com©.

#### **8. CB1R and adult spinal cord neurogenesis**

Taking the adult brain as an example of endocannabinoid system involvement in progenitor cell proliferation and differentiation, there is a possibility that the spinal cord progenitors may also be modulated by this system. There is an overwhelming lack of published studies addressing the presence and roles of the endocannabinoid system in adult spinal cord progenitor cells. Of particular importance is that CB1R is widely distributed on cells throughout the spinal cord, but also in lamina X, which includes the putative progenitor cell niche. We have identified CB1R on adult spinal cord-derived Nestin(+) progenitor cells in primary cultures (**Figure 7**).

Fig. 6. Transverse section of an adult mouse spinal cord, depicting a model for progenitor cell and immature neuron location. Based on the work by Shechter et al, 2007, 2011, the majority of the GABAergic, BrdU(+)/ DCX(+) immature neurons reside in the gray matter of the dorsal horn. Under physiological conditions, the levels of these cells depend on the type of and exposure to sensory environmental enrichment. Image generated with

**Progenitor cells** 

**DCX + Immature Neurons** 

Taking the adult brain as an example of endocannabinoid system involvement in progenitor cell proliferation and differentiation, there is a possibility that the spinal cord progenitors may also be modulated by this system. There is an overwhelming lack of published studies addressing the presence and roles of the endocannabinoid system in adult spinal cord progenitor cells. Of particular importance is that CB1R is widely distributed on cells throughout the spinal cord, but also in lamina X, which includes the putative progenitor cell niche. We have identified CB1R on adult spinal cord-derived Nestin(+) progenitor cells in

motifolio.com©.

primary cultures (**Figure 7**).

**8. CB1R and adult spinal cord neurogenesis** 

Fig. 7. Primary adult spinal cord cultures from rats contain Nestin(+) progenitor cells (red), which also express CB1R (green). The role of CB1R on these progenitors has not been examined, and further studies are needed to determine how the receptor is involved in progenitor cell quiescence, proliferation or differentiation. Image obtained after 6 days *in vitro* with 63X objective.

In response to injury, not only do progenitor cells proliferate in the spinal cord (Frisen, Johansson et al. 1995; Johansson, Momma et al. 1999; Namiki and Tator 1999; Shibuya, Miyamoto et al. 2002), but levels of endocannabinoids, receptors and enzymes are also altered as described earlier (in Section 4). Rigorous studies are needed to address if and how adult spinal cord progenitor cells respond to endogenous cannabinoid tone or to exogenously administered cannabinoids. Does endocannabinoid tone contribute to the nonneurogenic spinal cord environment? Are endo/exo-cannabinoids capable of promoting spinal cord neurogenesis or gliogenesis? These are just a few critical and novel avenues for potentially promoting neurogenesis in the adult spinal cord.

#### **9. The effect of chronic cannabinergic drug use on the CNS- implications for the treatment of chronic pain**

Cannabis is used both acutely and chronically for recreational or medicinal purposes. There is controversy regarding medical marijuana because of the documented cognitive side effects of chronic recreational use (Jager and Ramsey 2008; Hester, Nestor et al. 2009; Battisti, Roodenrys et al. 2010). However, all drugs come with a risk-benefit consideration, and a plethora of historical and emerging evidence indicates that the medicinal value of cannabis cannot be ignored. Many studies have demonstrated that endocannabinoids and application of exogenous cannabinoids (usually mixed CB1R/CB2R agonists) reduce pain sensation (Guindon and Hohmann 2009). Presently, such an approach is becoming more clinically accepted for treating chronic pain states (Aggarwal, Carter et al. 2009; Karst, Wippermann et al. 2010; Lynch and Campbell 2011). While CB2R activation attenuates nociception mostly by modulating the inflammatory response (Guindon and Hohmann 2008), the role of CB1R is more complex because its location on various cells along the pain pathways appears to contribute differently to nociception. Moreover, many cannabinergic drugs are not only mixed agonists, but may bind non-specifically to other receptors, including TRP-channels (Patwardhan, Jeske et al. 2006; Patil, Patwardhan et al. 2011).

The use of several CB1R knock-outs (global and conditional) has helped to clarify the role of these receptors in nociception. Recent worked demonstrated that cannabinoids mediate analgesia by activating CB1Rs located on peripheral nociceptors (dorsal root ganglia sensory neurons) (Agarwal, Pacher et al. 2007). Interestingly, by using *in vitro* spinal cord slices and *in vivo* recordings of dorsal horn neurons, activation of CB1Rs on spinal cord dorsal horn

The Cannabinoid 1 Receptor and Progenitor Cells in the Adult Central Nervous System 115

still needed to understand how the endocannabinoid system affects these cells normally and

Agarwal, N., P. Pacher, et al. (2007). "Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors." Nat Neurosci 10(7): 870-879. Aggarwal, S. K., G. T. Carter, et al. (2009). "Medicinal use of cannabis in the United States:

Aguado, T., K. Monory, et al. (2005). "The endocannabinoid system drives neural progenitor

Aguado, T., J. Palazuelos, et al. (2006). "The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor cells." J Neurosci 26(5): 1551-1561. Aldrich, M. R. (2006). "The Remarkable W.B. O'Shaughnessy" Retrieved January 29, 2011, from http://antiquecannabisbook.com/chap2B/Shaughnessy/Shaughnessy.htm. Arevalo-Martin, A., D. Garcia-Ovejero, et al. (2008). "CB2 cannabinoid receptors as an

Barnabe-Heider, F., C. Goritz, et al. (2010). "Origin of new glial cells in intact and injured

Battisti, R. A., S. Roodenrys, et al. (2010). "Chronic use of cannabis and poor neural efficiency in verbal memory ability." Psychopharmacology (Berl) 209(4): 319-330. Bisogno, T., F. Berrendero, et al. (1999). "Brain regional distribution of endocannabinoids:

Bisogno, T. and V. Di Marzo (2010). "Cannabinoid receptors and endocannabinoids: role in

Bouaboula, M., C. Poinot-Chazel, et al. (1995). "Activation of mitogen-activated protein

Brown, S. P., P. K. Safo, et al. (2004). "Endocannabinoids inhibit transmission at granule cell

Carrier, E. J., C. S. Kearn, et al. (2004). "Cultured rat microglial cells synthesize the

Caulfield, M. P. and D. A. Brown (1992). "Cannabinoid receptor agonists inhibit Ca current

Costa, B., A. E. Trovato, et al. (2005). "Effect of the cannabinoid CB1 receptor antagonist,

receptor-dependent mechanism." Mol Pharmacol 65(4): 999-1007.

chronic constriction injury of the sciatic nerve." Pain 116(1-2): 52-61.

historical perspectives, current trends, and future directions." J Opioid Manag 5(3):

emerging target for demyelinating diseases: from neuroimmune interactions to cell

implications for their biosynthesis and biological function." Biochem Biophys Res

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kinases by stimulation of the central cannabinoid receptor CB1." Biochem J 312 ( Pt

to Purkinje cell synapses by modulating three types of presynaptic calcium

endocannabinoid 2-arachidonylglycerol, which increases proliferation via a CB2

in NG108-15 neuroblastoma cells via a pertussis toxin-sensitive mechanism." Br J

SR141716, on nociceptive response and nerve demyelination in rodents with

in response to injury and disease.

**11. References** 

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adult spinal cord." Cell Stem Cell 7(4): 470-482.

channels." J Neurosci 24(24): 5623-5631.

Commun 256(2): 377-380.

Pharmacol 106(2): 231-232.

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2): 637-641.

neurons actually enhances (not reduces) nociceptive responses (Pernia-Andrade, Kato et al. 2009; Zhang, Chen et al. 2010). Stimulation of spinal cord CB1Rs inhibits the release of GABA, glycine (Pernia-Andrade 2009), and opioids, while enhancing the release of substance P (Zhang, Chen et al. 2010). Therefore, CB1R activation may contribute to nociception by increasing excitability at the spinal cord level. Consequently, CB1R antagonists have shown anti-nociceptive efficacy in several experimental pain models (Costa, Trovato et al. 2005; Croci and Zarini 2007; Pernia-Andrade, Kato et al. 2009). On the contrary, another recent study also using *in vivo* recordings demonstrated that blocking spinal CB1Rs enhanced the evoked response of the spinal cord dorsal horn neurons in neuropathic rats, indicative of a pro-nociceptive role of the receptor (Sagar, Jhaveri et al. 2010). One explanation for these different results could be attributed to the anesthetic used. Pernia-Andrade et al 2009 used a mixture of pentobarbital with pancuronium (a muscle relaxant), while Sagar et al.'s study only used isoflurane. The use of a muscle relaxant would allow the use of lower levels of the anesthetic to achieve immobility (required for the *in vivo* recordings). It is possible that the level of anesthesia used in Sagar et al's 2010 recordings may have depressed the neuronal activity relevant to pain sensation. Consistent with this possibility is that there was no difference in the firing rate of dorsal horn neurons in anesthetized neuropathic and sham operated animals at various levels of stimulation.

The chronic use of mixed cannabinoid drugs should be further investigated in light of the fact that the majority readily cross the blood-brain barrier. These compounds may be capable of providing pain relief, but they may also be affecting other important cellular functions, such as neurogenesis in the brain and spinal cord. Neurogenesis from endogenous progenitor cells is associated with a wide range of functions, and perturbations of this process are correlated with disease symptoms. Interference of physiological neurogenesis may be a highly undesirable side-effect of chronic endocannabinoid system manipulation by the use of CB1R/CB2R agonists or antagonists. For example, following peripheral nerve injury or direct spinal cord injury, a specific loss of inhibitory GABAergic interneurons in the spinal cord dorsal horn is postulated to be a major contributor to chronic pain (Moore, Kohno et al. 2002; Scholz, Broom et al. 2005; Meisner, Marsh et al. 2010). Replacement of these neurons through neurogenesis is an attractive therapeutic strategy because it attempts to go beyond the management of symptoms; it targets an underlying biological phenomenon of neuronal death following injury. Given the controversy regarding how cannabinoids modulate neurogenesis, it is possible that while treatment with mixed cannabinoids can ameliorate pain, long term usage may prevent the replacement of damaged inhibitory neurons by blocking neurogenesis, and thus contribute to an underlying etiology of chronic pain. Hence understading the role of the individual CBRs in adult neurogenesis, but also during pain states, could help discern how to more susccesfully use these agents clinically.

#### **10. Conclusions**

CB1R expression on adult CNS-derived progenitor cells is not only indicative of endogenous cannabinoid modulation, but also points to potential consequences of cannabinoid pharmacotherapy on progenitor proliferation and differentiation- whether beneficial or deleterious. The complex results published about adult brain progenitors and the lack of data on adult spinal cord progenitors demonstrate that extensive basic research is still needed to understand how the endocannabinoid system affects these cells normally and in response to injury and disease.

#### **11. References**

114 Pharmacology

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The chronic use of mixed cannabinoid drugs should be further investigated in light of the fact that the majority readily cross the blood-brain barrier. These compounds may be capable of providing pain relief, but they may also be affecting other important cellular functions, such as neurogenesis in the brain and spinal cord. Neurogenesis from endogenous progenitor cells is associated with a wide range of functions, and perturbations of this process are correlated with disease symptoms. Interference of physiological neurogenesis may be a highly undesirable side-effect of chronic endocannabinoid system manipulation by the use of CB1R/CB2R agonists or antagonists. For example, following peripheral nerve injury or direct spinal cord injury, a specific loss of inhibitory GABAergic interneurons in the spinal cord dorsal horn is postulated to be a major contributor to chronic pain (Moore, Kohno et al. 2002; Scholz, Broom et al. 2005; Meisner, Marsh et al. 2010). Replacement of these neurons through neurogenesis is an attractive therapeutic strategy because it attempts to go beyond the management of symptoms; it targets an underlying biological phenomenon of neuronal death following injury. Given the controversy regarding how cannabinoids modulate neurogenesis, it is possible that while treatment with mixed cannabinoids can ameliorate pain, long term usage may prevent the replacement of damaged inhibitory neurons by blocking neurogenesis, and thus contribute to an underlying etiology of chronic pain. Hence understading the role of the individual CBRs in adult neurogenesis, but also during pain states, could help discern how to more susccesfully

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**6** 

*Canada* 

**Peroxisome Proliferator Activated** 

*Université de Sherbrooke, Research Center on Aging* 

Jennifer Tremblay-Mercier

**Receptor Alpha (PPAR) Agonists:** 

**A Potential Tool for a Healthy Aging Brain** 

Cognitive decline related to advancing age includes many sub-categories of diseases, some more or less well defined and understood. First, there is "normal" cognitive decline, which is gradual and progressive during aging and seems inevitable. When cognitive decline is large enough to disrupt the activities of daily life, a state of dementia is diagnosed. There are several types of dementia according to the etiology of cognitive decline: vascular dementia, which results from a circulatory disorder causing an obstruction of cerebral blood vessels which leads to the progressive degeneration of brain cells due to a lack of oxygen. Vascular dementia represents 20% of all cases of dementia. Lewis body dementia is an accumulation of α-synuclein protein within the cell and it represents 5 to 15% of neurodegenerative diseases. Frontotemporal dementia as the name suggests, is a degeneration of the region of the frontal and temporal anterior cortex. The reasons for this degeneration are not fully understood. Alzheimer's disease (AD) represents the majority of cases of dementia (65%)

The most accepted theory in the medical community to explain the origin of AD is currently the accumulation of β-amyloid protein in the form of plaques accompanied by neurofibrillary tangles of tau protein that cause neuronal death and loss of brain matter. However, this theory is challenged for many reasons. The high profile failures of antiamyloid interventions and lack of agreement on which form the β-amyloid is toxic and the mechanism by which this occurs force the scientific community to consider amyloid only as one part of a multi-factorial disease process including a variety of aggravating factors. A recent paper entitled "Changing perspectives on Alzheimer's Disease: Thinking outside the

The clinical diagnosis of AD is based on clinical examination and confirmed by neuropsychological tests and is diagnosed through exclusion. That means if the person

although its etiology is not known exactly, or rather multi-factorial.

amyloid Box" resume this thinking (D'Alton & George, 2011).

**1.2 Alzheimer's disease diagnosis** 

**1. Introduction** 

**1.1 Definitions and considerations** 

