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Distribution of the Endocannabinoid
System in the Central Nervous System
Sherry Shu-Jung Hu and Ken Mackie
Contents
1 Introduction ................................................................................... 61
1.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 61
1.2 Cells Expressing Components of the ECS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
1.3 Subcellular Localization of CB
1
Cannabinoid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2 Retina ......................................................................................... 64
2.1 Receptors . . ............................................................................. 64
2.2 Synthetic Enzymes . .................................................................... 64
2.3 Degradative Enzymes . . . ............................................................... 64
3 Cerebral Cortex.............................................................................. 65
3.1 Neocortex ............................................................................... 65
3.1.1 Receptors . . ..................................................................... 65
3.1.2 Synthetic Enzymes ............................................................. 66
3.1.3 Degradative Enzymes .......................................................... 66
3.2 Olfactory Areas (Olfactory Bulb, Piriform Cortex, Associated Regions) . . . . . . . . . . . 68
3.2.1 Receptors . . ..................................................................... 68
3.2.2 Synthetic Enzymes ............................................................. 68
3.2.3 Degradative Enzymes .......................................................... 68
3.3 Hippocampal Formation . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.3.1 Receptors . . ..................................................................... 69
3.3.2 Synthetic Enzymes ............................................................. 71
3.3.3 Degradative Enzymes .......................................................... 71
S.S.-J. Hu
Department of Psychology, National Cheng Kung University, Tainan 70101, Taiwan
e-mail: shujunghu@gmail.com
K. Mackie (*)
Department of Psychological and Brain Sciences, Indiana University, 47405 Bloomington, IN,
USA
Gill Center for Biomolecular Science, Indiana University, 47405 Bloomington, IN, USA
e-mail: kmackie@indiana.edu
#Springer International Publishing Switzerland 2015
R.G. Pertwee (ed.), Endocannabinoids, Handbook of Experimental Pharmacology
231, DOI 10.1007/978-3-319-20825-1_3
59
3.4 Cortical Subplate (Other Amygdala Nuclei) . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 73
3.4.1 Receptors . . ..................................................................... 73
3.4.2 Synthetic Enzymes ............................................................. 73
3.4.3 Degradative Enzymes .......................................................... 73
4 Subcortical Nuclei (Striatum, Basal Ganglia) . .............................................. 74
4.1 Striatum (Dorsal, Caudate) . . . . . . . . . . . . ................................................ 74
4.1.1 Receptors . . ..................................................................... 74
4.1.2 Synthetic Enzymes ............................................................. 74
4.1.3 Degradative Enzymes .......................................................... 75
4.2 Striatum (Ventral, Accumbens) ....................................................... 75
4.2.1 Receptors . . ..................................................................... 75
4.2.2 Synthetic Enzymes ............................................................. 75
4.2.3 Degradative Enzymes .......................................................... 75
4.3 Striatum Medial (Lateral Septum, Septohippocampal, etc.) .......................... 76
4.3.1 Receptors . . ..................................................................... 76
4.3.2 Synthetic Enzymes ............................................................. 76
4.3.3 Degradative Enzymes .......................................................... 76
4.4 Striatum Caudal (Striatum-like Amygdala Nuclei, Central Amygdala, Bed Nucleus,
Medial Amygdala, Etc.) . . . . ........................................................... 76
4.4.1 Receptors . . ..................................................................... 76
5 Cerebellum and Associated Nuclei . ......................................................... 77
5.1 Cerebellar Cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.1.1 Receptors . . ..................................................................... 77
5.1.2 Synthetic Enzymes ............................................................. 77
5.1.3 Degradative Enzymes .......................................................... 78
5.2 Deep Cerebellar Nuclei (Fastigial, Interpos, Dentate Nucleus) . . . . . . . . . . ............ 78
5.2.1 Receptors . . ..................................................................... 78
5.2.2 Synthetic Enzymes ............................................................. 78
5.2.3 Degradative Enzymes .......................................................... 78
6 Brainstem ..................................................................................... 79
6.1 Diencephalon . .......................................................................... 79
6.1.1 Thalamus (All Nuclei, Including Reticular Thalamic Nucleus, Habenula) .. 79
6.1.2 Hypothalamus (All Nuclei) .................................................... 80
6.1.3 Mesencephalon (Colliculi, VTA, PAG, SN, Raphe) . ........................ 80
6.2 Hindbrain ............................................................................... 82
6.2.1 Medulla (Area Postrema, Cochlear Nuclei, Nucleus of the Solitary Tract,
Trigeminal Nuclei, Various Other Cranial Nerve Nuclei) . . . . . . . . . . . . . . . . . . . 82
7 Spinal Cord (Dorsal, Ventral, Dorsal Root Ganglion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
7.1 Receptors . . ............................................................................. 83
7.2 Synthetic Enzymes . .................................................................... 84
7.3 Degradative Enzymes . . . ............................................................... 85
8 Summary, Concluding Thoughts, and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Abstract
The endocannabinoid system consists of endogenous cannabinoids
(endocannabinoids), the enzymes that synthesize and degrade endocannabinoids,
and the receptors that transduce the effects of endocannabinoids. Much of what
we know about the function of endocannabinoids comes from studies that
combine localization of endocannabinoid system components with physiological
60 S.S.-J. Hu and K. Mackie
or behavioral approaches. This review will focus on the localization of the best-
known components of the endocannabinoid system for which the strongest
anatomical evidence exists.
Keywords
CB
1
cannabinoid receptor • Immunocytochemistry • In situ hybridization
Abbreviations
2-AG 2-Arachidonoylglycerol
ABHD4/6/12 α/β-hydrolase domain 4/6/12
AEA or anandamide N-arachidonoylethanolamine
BLA Basolateral amygdala
CB
1
and CB
2
Cannabinoid receptor 1 and 2
CRIP1a Cannabinoid receptor interacting protein 1a
DAGLα/βDiacylglycerol lipase α/β
DRG Dorsal root ganglion
FAAH Fatty acid amide hydrolase
GDE1 Glycerophosphodiesterase 1
M1 Muscarinic cholinergic receptor
MAGL Monoacylglycerol lipase
mGluR5 Metabotropic glutamate receptor 5
MSNs Striatal medium spiny neurons
NAAA N-acylethanolamine-hydrolyzing acid amidase
NAPE-PLD N-acyl phosphatidylethanolamine phospholipase D
PAG Periaqueductal gray
SN Substantia nigra
TH Tyrosine hydroxylase
VTA Ventral tegmental area
1 Introduction
1.1 Overview
Studies of the distribution of the protein components of the endocannabinoid
system (ECS) are motivated by the notion that we can gain important insights
into the function of the ECS by understanding the location of its component
enzymes and receptors. This review has been written with that concept in mind,
with an emphasis on studies that integrate function and location. Because of space
limitations, this review will focus on components of the ECS in the CNS with the
highest quality localization data. Thus, the emphasis will be on the cannabinoid
Distribution of the Endocannabinoid System in the Central Nervous System 61
CB
1
receptors and on the enzymes N-acyl phosphatidylethanolamine phospholipase
D (NAPE-PLD) (Di Marzo et al. 1994; Okamoto et al. 2004), fatty acid amide
hydrolase (FAAH) (Cravatt et al. 2001), diacylglycerol lipase (DAGL) (Bisogno
et al. 2003; Gao et al. 2010), monoacylglycerol lipase (MAGL) (Dinh et al. 2002,
2004), and α/ß-hydrolase domain 6 (ABHD6) (Blankman et al. 2007; Marrs
et al. 2010). The interesting topic of neuronal expression of CB
2
receptors is
complicated by the high inducibility of CB
2
in pathological conditions, the low
levels of CB
2
compared to CB
1
, the presence of CB
2
in microglia and endothelial
cells, and nonspecific antibodies. Nonetheless, CB
2
may be present on a limited
population of neurons with functional consequences, for example: (Viscomi
et al. 2009; den Boon et al. 2012; Zhang et al. 2014). The interested reader can
refer to the literature for additional details and consideration (Marsicano and Kuner
2008; Atwood and Mackie 2010). We will also focus on ECS component localiza-
tion in the mature brain in this review. The ECS and its components are subject to
dynamic regulation in the developing CNS, a topic that has been well covered in
recent reviews (Harkany et al. 2008; Maccarrone et al. 2014).
A variety of techniques are available to determine protein localization in tissues.
These include autoradiography (labeled ligands or GTPγS), in situ hybridization,
and antibody-based techniques. Each of these techniques gives complementary
information, which taken together, can enhance our understanding of ECS func-
tion—autoradiography requires high affinity binding of a probe to the protein,
in situ hybridization detects mRNA (often useful for identifying cell types
synthesizing a protein), and antibody-based techniques detect a (protein) epitope
resembling the epitope that antibody was raised against. As antibody-based
techniques are most commonly used for ECS localization, it is important to be
aware of some of their caveats. The issue of spurious results from antibody studies
has received considerable attention in the neuroscience community (Saper 2005;
Rhodes and Trimmer 2006; Michel et al. 2009; Manning et al. 2012), though
numerous examples of poor practice continue to be published. “Best practices”
have been discussed and implemented by several journals (Saper 2005; Rhodes and
Trimmer 2006; Manning et al. 2012) and are summarized in Table 1. As much as is
possible, studies that have adhered to those practices will be emphasized in this
review.
1.2 Cells Expressing Components of the ECS
While the ECS in neurons has received the most attention due to the prominent
effects of endogenous and exogenous cannabinoids on neuronal function, it is
important to appreciate that glial cells are a major component of the ECS, some-
times acting independently of neurons and sometimes in concert. Strong evidence
supports the presence of CB
1
receptors in some astrocytes and microglia
(Rodriguez et al. 2001; Stella 2010; Bosier et al. 2013), and these cells as well as
oligodendrocytes are prodigious synthesizers and degraders of endocannabinoids
(Walter et al. 2002; Stella 2009); however, their complement of enzymes vary
somewhat from those in neurons (Marrs et al. 2010). In addition, the CNS
62 S.S.-J. Hu and K. Mackie
vasculature also participates in endocannabinoid signaling (Gebremedhin
et al. 1999; Schley et al. 2009; Zhang et al. 2009; Dowie et al. 2014).
1.3 Subcellular Localization of CB
1
Cannabinoid Receptors
Due to their prominent effects on presynaptic calcium channels and synaptic
transmission, it is not surprising that high levels of CB
1
receptors are found on
some presynaptic terminals and preterminal axon segments (Katona et al. 1999;
Nyiri et al. 2005a). The ability of endocannabinoids to suppress spiking in low
threshold spiking cortical interneurons and some pyramidal cells suggests that CB
1
receptors are also located on neuron somata (Marinelli et al. 2009). Within neurons,
Table 1 Antibody controls for immunocytochemistry
Approach Strength
Block with immunizing protein Weak (block with immunizing protein is a necessary,
but not sufficient condition to establish antibody
specificity)
Lack of staining with preimmune
serum
Weak (immune response can induce the expression of
many serum proteins that can interact with extraneous
epitopes)
Lack of staining in knockout (KO) Strong (requires knockout; need to understand how
the KO was made, articularly if the exon which the
antibody was raised against remains; knockout
studies should be conducted in parallel (identical
tissue processing, incubation times, etc.) with
experiments performed with tissue that expresses the
genetically deleted protein
Identical staining with antibody
directed against independent epitopes
Strong (for alternatively spliced proteins need to
ascertain that the appropriate exons are expressed)
Lack of staining in knockdown Strong (need independent verification of knockdown;
stronger if knockdown from limited population of
cells so “controls” are adjacent)
Detection of protein in transfected cells Medium (demonstrates antibody can detect protein,
but not necessarily in tissue; stronger if conducted in
a mixed population of cells (expressing and
non-expressing) and target protein is epitope-tagged
to allow its unequivocal detection; difficult to apply if
target protein is present in the cell line used)
Detection of appropriate band on
western blot
Can be helpful correlation (however, the
conformation of protein in fixed tissue and denatured
gel is quite different; many examples where antibody
won’t detect a specific band in Western blots and
works for immunocytochemistry and vice versa)
Correlation with in situ hybridization Useful at the level of cell populations (assumes
mRNA is translated to protein)
Correlation with function Helpful (e.g., CB
1
-mediated responses and CB
1
receptors detected; however can lead to circular
reasoning)
Distribution of the Endocannabinoid System in the Central Nervous System 63
CB
1
receptors are sometimes associated with specialized structures (e.g., multi-
lamellar bodies (Katona et al. 1999)) that may be involved in their trafficking. A
recent though not uncontroversial finding (Benard et al. 2012; Hebert-Chatelain
et al. 2014a,b; vs. Morozov et al. 2013) is that CB
1
receptors are associated with
some mitochondria, including mitochondria found in astrocytes, where they con-
tribute to energy balance and may play a role in synaptic plasticity.
2 Retina
2.1 Receptors
Using the immunocytochemical approach, CB
1
receptors are found in subsets of
amacrine cells and horizontal cells and densely expressed in the inner plexiform
layer (Straiker et al. 1999a; Yazulla et al. 1999). CB
1
are also present in rod and
cone photoreceptor terminals in a wide range of vertebrate retinas, including human
(Straiker et al. 1999a,b; Hu et al. 2010), as well as in rod bipolar cells in rat retina
(Yazulla et al. 1999).
2.2 Synthetic Enzymes
While the presence of synthetic enzymes for N-arachidonoylethanolamine (anan-
damide or AEA) has not yet been examined in retina, two isoforms of the major
synthetic enzyme for 2-arachidonoylglycerol (2-AG), diacylglycerol lipase-αand -
β(DAGLα/β) (Bisogno et al. 2003; Gao et al. 2010), were both found in the mouse
retina. DAGLαappears in the two synaptic layers, the outer plexiform layer and
inner plexiform layer, whereas DAGLβimmunoreactivity is limited to retinal blood
vessels. Furthermore, DAGLαis present in postsynaptic type 1 OFF cone bipolar
cells juxtaposed to CB
1
-containing cone photoreceptor terminals (Hu et al. 2010).
These findings suggest that retrograde 2-AG signaling exists at type 1 OFF bipolar
cell-cone photoreceptor synapses, consisting of presynaptic CB
1
receptors and
postsynaptic DAGLα.
2.3 Degradative Enzymes
Both degradative enzymes for anandamide and related N-acylethanolamines
(NAEs), fatty acid amide hydrolase (FAAH) (Cravatt et al. 2001) and N-acyletha-
nolamine-hydrolyzing acid amidase (NAAA) (Guo et al. 2005; Tsuboi et al. 2005),
are present in retina. FAAH immunoreactivity was first detected in large ganglion
cells, in large dopaminergic amacrine cells, in the dendrites of star-burst amacrine
cells, and in the somata of horizontal cells of the rat retina (Yazulla et al. 1999). In
the mouse retina, FAAH is widely expressed in the inner segments, outer nuclear
layer, ganglion cell layer, and in the axon terminals of photoreceptors in the outer
plexiform layer, and also co-localizes with CB
1
in subpopulation of amacrine cells
64 S.S.-J. Hu and K. Mackie
in the inner nuclear layer and in cells in the ganglion cell layer (Hu et al. 2010). The
most notable difference between mouse and rat FAAH staining is the absence of
staining in horizontal cells of the mouse, possibly a function of species difference.
Finally, NAAA immunoreactivity is limited to retinal pigment epithelium in the
mouse retina (Hu et al. 2010).
Among five candidate degradative enzymes for 2-AG, monoacylglycerol lipase
(MAGL) and α/β-hydrolase domain 6 (ABHD6) (Blankman et al. 2007) were found
in the mouse retina. Similar to DAGLα, MAGL staining was found in the outer and
inner plexiform layers and additionally in the ganglion cell layer (Hu et al. 2010).
While MAGL is not co-localized with DAGLαin the inner plexiform layer, it is
distal to DAGLαin the outer plexiform layer. MAGL is present in photoreceptor
terminals, including the rod spherules and cone pedicles (Hu et al. 2010), where it is
well positioned to break down 2-AG after retrograde release onto both rod and cone
terminals.
On the other hand, ABHD6 is widely distributed in the inner plexiform layer,
inner nuclear layer, and ganglion cell layer. ABHD6 is localized to the calbindin-
and GAD67-positive amacrine cells in the inner nuclear layer and to the dendrites
of ganglion or displaced amacrine cells in the proximal inner plexiform layer
(Hu et al. 2010). The postsynaptic staining of ABHD6 suggests its potential role
in the breakdown of extrasynaptic 2-AG that has diffused beyond its intended target
synapses.
3 Cerebral Cortex
3.1 Neocortex
3.1.1 Receptors
CB
1
receptors are densely expressed in all regions of the cortex, with high levels
found in cingulate gyrus, frontal cortex, secondary somatosensory, and motor
cortex, reviewed in (Mackie 2005). An immunocytochemical study found a hetero-
geneous distribution of CB
1
-immunoreactive axons across neocortex in macaque
monkeys and humans. Most neocortical association regions, such as the prefrontal
and cingulate cortex, contain a higher density of CB
1
-immunoreactive axons
compared to the primary motor and somatosensory cortices (Eggan and Lewis
2007). Furthermore, in many cortical regions, CB
1
-immunoreactivity displays a
distinctly laminar pattern of expression, corresponding to the cytoarchitectonic
boundaries. Although the distribution of CB
1
immunoreactivity across monkey
neocortical regions is broadly similar to that observed in the rat using immunocy-
tochemical (Egertova and Elphick 2000; Hajos et al. 2000; Katona et al. 2001;
Bodor et al. 2005) and autoradiographic (Herkenham et al. 1991) approaches, there
are several species differences in the laminar distribution of CB
1
-immunoreactive
axons. For example, CB
1
-immunoreactive axons were reported to be most densely
expressed in layers 2–3 and 6 and least densely expressed in layer 4 of the rat frontal
and cingulate cortex (Egertova and Elphick 2000). In contrast, the highest density
of CB
1
-immunoreactive axons is localized in layer 4 of the same regions in monkey
Distribution of the Endocannabinoid System in the Central Nervous System 65
(Eggan and Lewis 2007). In the primary somatosensory cortex, CB
1
-immunoreac-
tive axons are most densely localized in layer 5A in rat (Bodor et al. 2005), whereas
a relatively similar density of CB
1
-immunoreactive axons exists in layers 2–3 and
5–6, and a sparse axonal labeling presents in layer 4 of monkey (Eggan and Lewis
2007). Despite these differences, the laminar distribution of CB
1
-immunoreactivity
is quite similar within primates. For example, both autoradiographic (Glass
et al. 1997) and immunocytochemical (Eggan and Lewis 2007) methods yield
similar laminar distribution of CB
1
receptors in human and monkey neocortex,
respectively.
In forebrain, high levels of CB
1
receptors have been primarily found on large
cholecystokinin (CKK)-containing basket interneurons, with lesser levels found in
non-CCK-expressing neurons (Marsicano and Lutz 1999). For example, CB
1
immunoreactivity is absent in nonadapting multipolar interneurons, such as the
parvalbumin or bi-tufted adapting somatostatin-expressing interneurons (Tsou
et al. 1999; Bodor et al. 2005). Despite original studies suggesting lack of expres-
sion of CB
1
immunoreactivity in principal glutamatergic neurons (Tsou
et al. 1998a; Freund et al. 2003), more sensitive in situ hybridization studies
revealed low but detectable levels of CB
1
mRNA in the great majority of
glutamatergic neurons in many cortical regions including neocortex (Monory
et al. 2006). Moreover, a single-cell real-time polymerase chain reaction (qPCR)
study revealed that at least 50% of neocortical glutamatergic pyramidal neurons
contain CB
1
mRNA (Hill et al. 2007), which is consistent with functional data
showing that the CB
1
receptor agonist WIN-55212-2 decreased the intracortical
electrical stimulation-evoked excitatory postsynaptic currents (EPSCs) in a CB
1
antagonist-dependent fashion (Hill et al. 2007). In summary, CB
1
receptors are
densely expressed in multiple cortical regions. While CB
1
expression is highest on
CCK-positive interneurons, functionally important CB
1
receptors are present on
multiple neuron populations.
3.1.2 Synthetic Enzymes
Studies examining the protein and mRNA distribution of the anandamide
synthesizing enzymes, NAPE-PLD, in neocortex have found it to be widespread,
but at relatively low levels (Egertova et al. 2008). On the other hand, in situ
hybridization revealed that moderate levels of DAGLαand DAGLβmRNA are
expressed in mouse cerebral cortex (Yoshida et al. 2006). DAGLαis typically
dendritic, as shown for a cultured cortical neuron in Fig. 1.
3.1.3 Degradative Enzymes
FAAH-immunoreactive neuronal somata and dendrites are present throughout the
transitional and neocortical regions of the mouse cerebral cortex and are often
surrounded by CB
1
-immunoreactive fibers. Except for layer 1, FAAH
immunostaining is evident in all cortical layers, especially in the large cells in
layer 5 (Egertova et al. 2003).
In situ hybridization revealed that high levels of MAGL mRNA exist throughout
the rat brain cortex, especially in layers 4, deep 5, and 6 (Dinh et al. 2002).
66 S.S.-J. Hu and K. Mackie
Fig. 1 Postsynaptic expression of diacylglycerol lipase alpha (DAGLα). (A) Dual immunofluo-
rescent staining for DAGLα(green) and MAP2 (red). DAGLαas detected by a C-terminal
antibody in a cultured neuron was found in close proximity to many dendritic spines. (Band C)
Two consecutive ultrathin sections from mouse spinal cord demonstrate that the electron-dense
reaction product representing DAGLαimmunoreactivity (arrowheads) is present in the dendrite
(d) close to the asymmetric postsynaptic density across from an excitatory terminal (b). Scale
bar ¼200 nm. Original figures provided by Barna Dudok and Istvan Katona (A) and Rita Nyilas
and Istvan Katona (B,C)
Distribution of the Endocannabinoid System in the Central Nervous System 67
In the mouse prefrontal cortex, ABHD6 immunoreactivity is predominantly
localized to the postsynaptic dendritic spines, which is juxtaposed to the CB
1
-
positive presynaptic terminals (Marrs et al. 2010). Moreover, selective inhibition
of ABHD6 allowed the induction of CB
1
-mediated long-term depression by the
subthreshold stimulation, suggesting this enzyme is a bona fide member of the
endocannabinoid signaling system (Marrs et al. 2010).
3.2 Olfactory Areas (Olfactory Bulb, Piriform Cortex, Associated
Regions)
3.2.1 Receptors
In the olfactory bulb, CB
1
receptors are highly expressed in the inner granular cell
layer, followed by the inner plexiform layer, while less are expressed in the external
plexiform layer, the glomerular layer, and the accessory olfactory bulb (Herkenham
et al. 1991; Tsou et al. 1998a; Egertova and Elphick 2000). However, a detailed
examination revealed that CB
1
receptor immunoreactivity is abundant in the
periglomerular processes of GAD65-positive interneurons and the inner granular
cell layer (A. Straiker, personal communication). Furthermore, CB
1
receptors are
expressed uniformly by most neurons in the anterior olfactory nucleus and the
anterior commissure, which connect the olfactory bulbs (Herkenham et al. 1991;
Matsuda et al. 1993; Glass et al. 1997; Tsou et al. 1998a; Egertova and Elphick
2000). Moreover, most neurons in the piriform cortex contain CB
1
mRNA
(Marsicano and Lutz 1999). Finally, CB
1
receptor immunoreactivity is present on
dendritic processes in the olfactory epithelium of Xenopus laevis tadpoles, where it
mediates cannabinoid modulation of odor-induced spike-associated currents in
individual olfactory receptor neurons (Czesnik et al. 2007).
3.2.2 Synthetic Enzymes
NAPE-PLD mRNA expression has been detected in several olfactory areas. For
example, NAPE-PLD mRNA is present in granule and periglomerular cells in the
olfactory bulb and in neuronal cell body layers in the olfactory tubercle and the
piriform cortex (Egertova et al. 2008). Interestingly, the immunostaining of NAPE-
PLD is very intense in glomeruli of the accessory olfactory bulb and in the
vomeronasal axons projecting into the accessory olfactory bulb (Egertova
et al. 2008). Moderate expression of DAGLαand DAGLβmRNA was found in
mouse olfactory bulb by in situ hybridization (Yoshida et al. 2006).
3.2.3 Degradative Enzymes
Despite the overall low levels of CB
1
receptor expression in the olfactory bulb,
FAAH immunoreactivity is intense, especially in fibers of the olfactory nerve and in
the olfactory glomeruli, as well as in the somata and dendrites of mitral cells
(Egertova et al. 2003). Moreover, FAAH-immunoreactive neuronal somata are
evident in the majority of cortical olfactory regions receiving direct input from
the olfactory bulb, including the anterior olfactory nucleus, the piriform cortex,
68 S.S.-J. Hu and K. Mackie
the tenia tecta, and the indusium griseum (Egertova et al. 2003). Importantly, in
all of these regions, FAAH-immunoreactive postsynaptic neuronal somata are
surrounded by a complementary network of CB
1
-immunoreactive fibers, which
supports the hypothesis that anandamide and other acyl amides may function as
transynaptic signaling molecules (Egertova et al. 2000).
3.3 Hippocampal Formation
3.3.1 Receptors
The hippocampus is highly involved in cognitive functions such as the spatial and
declarative learning and memory, thereby drawing much attention as a site of action
of endogenous and exogenous cannabinoids due to their effects on memory. Early
autoradiographic studies found very high levels of CB
1
receptors in all subfields of
the hippocampus as well as the dentate gyrus (Herkenham et al. 1991; Jansen
et al. 1992). In situ hybridization studies revealed that most CB
1
receptor expres-
sion arose from a restricted subset of interneurons (Matsuda et al. 1990,1993;
Mailleux and Vanderhaeghen 1992). Immunocytochemical studies showed high
levels of CB
1
receptors on large CKK-positive basket and Schaffer collateral-
associated interneurons in the hippocampal pyramidal cell layer, as well as the
molecular layer and at the base of the granule cell layer in the dentate gyrus (Katona
et al. 1999; Marsicano and Lutz 1999; Tsou et al. 1999; Egertova and Elphick 2000)
(e.g., Fig. 2a), while few or no CB
1
receptors were found on parvalbumin-positive
interneurons. Agonist activation of CB
1
receptors on these interneurons was found
to decrease power in theta, gamma, and ripple oscillations in the hippocampus
(Robbe et al. 2006). Actions of cannabinoids such as these appear to be widespread
in cortical and subcortical circuits (Sales-Carbonell et al. 2013) and may contribute
to the effects of cannabinoids on memory (Chen et al. 2003; Freund et al. 2003;
Klausberger et al. 2005; Robbe et al. 2006).
Building on earlier studies that detected CB
1
mRNA, but not protein in CA1 and
CA3 pyramidal neurons, studies in which CB
1
receptors were selectively deleted
from either GABAergic or glutamatergic neurons allowed the conclusive
anatomical identification of low levels of CB
1
protein in glutamatergic hippocam-
pal pyramidal neurons (Marsicano et al. 2003;Lutz2004). Indeed, several groups
independently identified CB
1
protein in glutamatergic pyramidal neurons in the
CA1 and CA3 regions (Degroot et al. 2006; Katona et al. 2006) (examples of CB
1
expression in hippocampal inhibitory and excitatory terminals are shown in
Fig. 2A–D). However, among glutamatergic neurons, the highest CB
1
levels are
present in dentate gyrus mossy cells (Kawamura et al. 2006; Monory et al. 2006).
Interestingly, many CB
1
-positive hilar mossy cells also contain dopamine D
2
receptors, suggesting that this region might be involved in the interactions of
these two neuromodulatory systems (Degroot et al. 2006). Moreover, CB
1
immu-
noreactivity was also identified in the majority of hippocampal cholinergic nerve
terminals, where presynaptic CB
1
receptors control acetylcholine release in vitro
(Gifford and Ashby 1996) and in vivo (Degroot et al. 2006). On the other hand, CB
1
Distribution of the Endocannabinoid System in the Central Nervous System 69
Fig. 2 CB
1
expression in rodent brain is primarily presynaptic. (A)CB
1
receptors in mouse
hippocampal formation were detected by an antibody directed to its C-terminus. A dense mesh-
work of CB
1
-expressing axons is evident. Levels are particularly high in the inner molecular layer
70 S.S.-J. Hu and K. Mackie
receptors appeared to be absent from granule cells of the dentate gyrus. Finally, low
but detectable levels of CB
1
receptors were found in a subset of progenitor cells in
the subgranular zone of the dentate gyrus, which regulate proliferation, survival,
and differentiation of these adult progenitor cells (Aguado et al. 2005; Galve-
Roperh et al. 2007).
3.3.2 Synthetic Enzymes
NAPE-PLD mRNA, as revealed by in situ hybridization, is most intensely
expressed in the dentate gyrus granule cell layer, followed by the pyramidal cell
layer of the hippocampus throughout all three fields (CA1-CA3) (Cristino
et al. 2008; Egertova et al. 2008; Nyilas et al. 2008). Moreover, NAPE-PLD
immunoreactivity was detected in granule cell axons (Egertova et al. 2008; Nyilas
et al. 2008) as well as in many neurons of the hilus region in mouse dentate gyrus
(Cristino et al. 2008; Nyilas et al. 2008). Therefore, in contrast to 2-AG’s prominent
role as a retrograde signaling messenger (Kano et al. 2009), anandamide and related
NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling
molecules to regulate the activity of postsynaptic neurons (Egertova et al. 2008).
High levels of DAGLαmRNA and protein have been found in postsynaptic
dendritic spines of the majority of hippocampal pyramidal neurons, including the
spine head, neck, or both (Katona et al. 2006; Yoshida et al. 2006). A similar pattern
for DAGLαwas found in the postmortem human hippocampus, with highest levels
in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum
moleculare of the dentate gyrus (Ludanyi et al. 2011). The juxtaposition of DAGL-
α-immunoreactive postsynaptic dendritic spines to CB
1
-expressing presynaptic
terminals at excitatory glutamatergic synapses highlights the prominence of 2-AG
as a retrograde messenger at many synapses (Katona et al. 2006).
3.3.3 Degradative Enzymes
FAAH is highly expressed in the somata and proximal dendrites of the pyramidal
cells, which are densely innervated by CB
1
-positive axon terminals (Egertova
et al. 1998,2003; Tsou et al. 1998b; Gulyas et al. 2004). However, it is important
to note that FAAH is more frequently present on the membrane surface of intracel-
lular organelles (e.g., mitochondria, smooth endoplasmic reticulum) than on
somatic or dendritic plasma membranes (Gulyas et al. 2004). In addition, FAAH
ä
Fig. 2 (continued) of the dentate gyrus and the pyramidal neuron layer: hi hilus, iml inner
molecular layer, oml outer molecular layer, pyr pyramidal cell layer, sr stratum radiatum, sub
subiculum. (B–D) Expression of CB
1
receptors at the ultrastructural level in the mouse hippocam-
pus assessed using a C-terminal CB
1
antibody. (B) Electron-dense reaction product is present in
both inhibitory (b
i
) and excitatory (b
e
) boutons. Note that the excitatory boutons synapse onto
spines (s) while the inhibitory boutons form synapses on a dendritic shaft. Dendritic structures are
pseudocolored blue for ease of identification. (C) Higher magnification image showing a CB
1
-
positive inhibitory terminal synapsing onto the shaft of a dendrite. (D) Higher magnification image
showing a CB
1
-positive excitatory bouton forming an asymmetric synapse onto a dendritic spine.
Scale bar ¼200 nm in B. Original figures provided by Jim Wager-Miller (A) and Chris Henstridge
and Istvan Katona (B–D)
Distribution of the Endocannabinoid System in the Central Nervous System 71
immunostaining is also evident in the somata of mouse dentate gyrus granule cells
(Egertova et al. 2003), which contrasts to the absence of FAAH immunoreactivity in
granule cells of the rat dentate gyrus (Tsou et al. 1998b; Gulyas et al. 2004). An in
situ hybridization study showed that MAGL mRNA is abundantly expressed in the
CA3 field of rat hippocampus (Dinh et al. 2002). Additional immunocytochemical
studies revealed that MAGL protein is particularly prominent in axon terminals of
granule cells, CA3 pyramidal cells, and some interneurons of rat hippocampus
(Gulyas et al. 2004), as well as in axon terminals of glutamatergic neurons in both
rodent and human hippocampus (Yoshida et al. 2006; Ludanyi et al. 2011) (Fig. 3A,
B).
Fig. 3 Presynaptic
localization of
monoacylglycerol lipase
(MAGL) detected with an
antibody recognizing residues
positioned in the middle of
mouse MAGL. (A) Punctate
expression of MAGL
immunoreactivity in the
mouse hippocampus
pyramidal cell layer. Scale
bar ¼10 μm. (B) Electron-
dense reaction product
representing MAGL
immunoreactivity is present
in an inhibitory terminal (b)
synapsing onto a dendritic
shaft (d). Original figures
provided by Stephen
Woodhams and Istvan Katona
72 S.S.-J. Hu and K. Mackie
3.4 Cortical Subplate (Other Amygdala Nuclei)
3.4.1 Receptors
The amygdala is divided into a cortical component (e.g., the basolateral, lateral, and
basomedial nuclei) and a striatal component (e.g., the central and medial nuclei)
(Swanson and Petrovich 1998). This subdivision corresponds to the different
structural organization and neurochemical properties of the two components. For
example, while most principal neurons in the cortical subdivision of the amygdala
are glutamatergic, the great majority of neurons in the striatal subdivision are
GABAergic. Therefore, similar to cortex, high levels of CB
1
receptors are primarily
expressed in the CCK-positive GABAergic basket cells (Marsicano and Lutz 1999;
Katona et al. 2001). For example, in the basal (but not lateral) nucleus of the
basolateral amygdala (BLA), high levels of CB
1
receptors are localized to presyn-
aptic CCK-positive GABAergic terminals at invaginating synapses (Yoshida
et al. 2011). On the other hand, as in cortex, low but functionally significant levels
of CB
1
receptor are present in glutamatergic neurons in the cortical part of amyg-
dala (Monory et al. 2006; Yoshida et al. 2011). Consistent with the above
anatomical data, functional studies suggest that CB
1
receptors and
endocannabinoids facilitated extinction of fear conditioning via inhibiting GABA
release in the BLA (Marsicano et al. 2002). Finally, in contrast to earlier studies
finding very weak CB
1
immunoreactivity within the central amygdala (Katona
et al. 2001; Kamprath et al. 2011), a recent study using a highly sensitive CB
1
receptor antibody showed the presence of functional CB
1
receptors in the central
amygdala (Ramikie et al. 2014).
3.4.2 Synthetic Enzymes
In the amygdaloid complex, NAPE-PLD mRNA is expressed in neurons of the
cortical and medial amygdaloid nuclei, while less staining has been found in the
basal and lateral nuclei (Egertova et al. 2008). At invaginating synapses, a unique
type of perisomatic synapses in the basal nucleus of the BLA, DAGLαis recruited
to somatic membrane of postsynaptic pyramidal neurons, juxtaposed to the CB
1
-,
MAGL-, and CCK-containing presynaptic terminals (Yoshida et al. 2011). In the
central amygdala, DAGLαis localized to postsynaptic dendritic spine heads and
dendritic shafts, juxtaposed to the CB
1
-containing presynaptic terminals at
glutamatergic synapses (Ramikie et al. 2014).
3.4.3 Degradative Enzymes
FAAH-immunoreactive neuronal somata are also present throughout the
basolateral complex of the amygdala, which includes the lateral, basolateral
(BLA), and basomedial nuclei. In all of these nuclei, the FAAH-immunoreactive
somata are surrounded by CB
1
-immunoreactive fibers (Egertova et al. 2003).
As mentioned above (Sect. 3.4.2), MAGL is co-expressed with CB
1
receptors in
the presynaptic CCK-positive terminals at invaginating synapses in the basal
nucleus of the BLA. Together with the postsynaptic DAGLαexpression, this
constitutes a molecular convergence for 2-AG-mediated retrograde signaling.
Distribution of the Endocannabinoid System in the Central Nervous System 73
This contrasts with the flat perisomatic synapses made by parvalbumin-positive
interneurons (Yoshida et al. 2011) where no such arrangement is present.
4 Subcortical Nuclei (Striatum, Basal Ganglia)
4.1 Striatum (Dorsal, Caudate)
4.1.1 Receptors
The subcortical nuclei with the highest level of CB
1
receptor expression are the
basal ganglia. In situ hybridization studies showed that many striatal medium spiny
neurons (MSNs) express CB
1
receptors, while adult pallidal and nigral neurons
contain little or no CB
1
mRNA (Matsuda et al. 1993; Julian et al. 2003). Due to its
axonal terminal localization, the high levels of pallidal and nigral CB
1
receptor
binding and protein observed in autoradiographic and immunocytochemical studies
mostly arose from GABAergic neurons projecting from the caudate putamen (Tsou
et al. 1998a; Egertova and Elphick 2000). The staining for CB
1
in axons is denser in
the globus pallidus than in the caudate putamen, while both show a gradient in the
staining intensity increasing from medial to lateral (Tsou et al. 1998a; Egertova and
Elphick 2000). Moreover, CB
1
receptors are present on both the striatonigral and
striatopallidal projection pathways; thus, they are well positioned to modulate both
the direct and indirect striatal output pathways (Hohmann and Herkenham 2000). In
addition, a study utilizing a high-sensitivity CB
1
receptor antibody showed CB
1
protein to be intensely expressed on GABAergic axon terminals of striatal MSNs
and parvalbumin-positive interneurons (Uchigashima et al. 2007). Finally, CB
1
protein was found on excitatory corticostriatal afferents (Gerdeman and Lovinger
2001; Rodriguez et al. 2001; Uchigashima et al. 2007), GABAergic aspiny
interneurons (Hohmann and Herkenham 2000) and neurons in the subthalamic
nucleus (Matsuda et al. 1993), with important functional implications (Kreitzer
and Malenka 2007).
4.1.2 Synthetic Enzymes
The immunostaining of NAPE-PLD is present in the caudate putamen (Egertova
et al. 2008). On the other hand, because 2-AG is synthesized by DAGL after
membrane depolarization and G
q
-coupled receptor activation, an immunocyto-
chemical study was carried out to examine the subcellular distribution of
DAGLα, metabotropic glutamate receptor 5 (mGluR5), and muscarinic cholinergic
receptor 1 (M1) in mouse striatum (Uchigashima et al. 2007). Even though all three
proteins were present on somatodendritic membranes of MSNs, only DAGLαand
mGluR5 were present in spines and the perisynaptic region, while M1 receptors
were absent in these domains (Uchigashima et al. 2007). This subcellular arrange-
ment may account for the differential involvement of mGluR5 and M1 in
endocannabinoid-mediated depolarization-induced suppression of inhibition and
depolarization-induced suppression of excitation (Uchigashima et al. 2007).
74 S.S.-J. Hu and K. Mackie
4.1.3 Degradative Enzymes
In the mouse caudate putamen, FAAH-immunoreactive oligodendrocytes are pres-
ent in fiber tracts (white matter) (Egertova et al. 2003). FAAH protein is also
localized to the myelin sheath surrounding the unstained axons of large neurons
(Egertova et al. 2003). This is consistent with the findings that FAAH mRNA is
present in white matter of the rat brain (Thomas et al. 1997). However, the
functional significance of FAAH expression in these non-neuronal cells is not yet
understood. Moreover, although FAAH and CB
1
receptors are anatomically
associated in many brain regions (Egertova et al. 1998), FAAH-expressing neurons
are present in some brain areas such as thalamus and midbrain that express few or
no CB
1
receptors (Egertova et al. 2003). Conversely, there is a population of striatal
GABAergic MSNs where CB
1
receptors are present in their axonal terminals
projecting to globus pallidus, entopeduncular nucleus, and substantia nigra, but
FAAH-expressing neurons are absent (Egertova et al. 2003). In these situations,
FAAH’s biological role may be to degrade related acyl amides other than ananda-
mide, for example, oleoylethanolamine and palmitoylethanolamine (Melis
et al. 2008).
4.2 Striatum (Ventral, Accumbens)
4.2.1 Receptors
CB
1
receptors are expressed at low to moderate levels in the nucleus accumbens,
with a pattern reminiscent of the striatum. CB
1
receptor protein is also localized on
the terminals of the prefrontal glutamatergic efferents projecting to the nucleus
accumbens as well as on the GABAergic axon terminals of accumbens MSNs and
parvalbumin-positive interneurons (Robbe et al. 2001; Uchigashima et al. 2007).
However, CB
1
receptors seem to be absent in the dopaminergic terminals projecting
from the ventral tegmental area (VTA) to the accumbens. Therefore, cannabinoid
stimulation of dopamine release in nucleus accumbens is likely mediated by
inhibition of GABA release (either from within the nucleus accumbens or in the
VTA) (Tanda et al. 1997; Szabo et al. 1999,2002).
4.2.2 Synthetic Enzymes
DAGLαimmunostaining, similar to that of mGluR5, is intense in spines and the
perisynaptic region of the somatodendritic surface of striatal MSNs (Uchigashima
et al. 2007).
4.2.3 Degradative Enzymes
FAAH immunoreactivity is absent in the nucleus accumbens (Egertova et al. 2003).
However, moderate amounts of MAGL mRNA were found in the nucleus
accumbens shell, islands of Calleja, and pontine nuclei by in situ hybridization
(Dinh et al. 2002).
Distribution of the Endocannabinoid System in the Central Nervous System 75
4.3 Striatum Medial (Lateral Septum, Septohippocampal, etc.)
4.3.1 Receptors
Moderate levels of CB
1
protein and mRNA are present in the basal forebrain
including the medial and lateral septum and the nucleus of the diagonal band
(Herkenham et al. 1991; Mailleux and Vanderhaeghen 1992; Matsuda et al. 1993;
Marsicano and Lutz 1999). In situ hybridization studies showed the presence of
CB
1
mRNA in cholinergic territories, especially the medial septum in mice. An
immunocytochemical study revealed that a dense network of CB
1
-positive fibers is
present in the tenia tecta, ventral pallidum, and substantia innominate, whereas a
fine network of CB
1
-positive fibers is localized to the medial septum, diagonal
bands, and nucleus basalis (Harkany et al. 2003). In the same study, no CB
1
immunoreactivity was detected in the cell bodies of basal forebrain cholinergic
cells; instead these cells contain high levels of FAAH (Harkany et al. 2003). This is
consistent with CB
1
receptors being synthesized and rapidly transported to axon
terminals. Indeed, a later study showed that CB
1
protein is expressed in at least
one-third of cholinergic neuron somata when axonal protein transport was blocked
by cholchicine (Nyiri et al. 2005b). In rat medial septum, at least two types of
cholinergic cells were identified, one with large somata, expressing CB
1
and
GABA
B
receptors and projecting to the hippocampus, whereas the other had
smaller somata and lacked these two receptors (Nyiri et al. 2005b). Therefore,
endocannabinoid signaling is implicated in the function of a population of septohip-
pocampal cholinergic neurons, including cognition as well as generation of hippo-
campal theta rhythms.
4.3.2 Synthetic Enzymes
The immunostaining of NAPE-PLD is localized to the lateral nucleus of the septum
(Egertova et al. 2008).
4.3.3 Degradative Enzymes
Neuronal FAAH-immunoreactivity has been detected in the lateral septum and the
triangular septal nucleus (Egertova et al. 2003), as well as in the basal forebrain
cholinergic neurons (Harkany et al. 2003).
4.4 Striatum Caudal (Striatum-like Amygdala Nuclei, Central
Amygdala, Bed Nucleus, Medial Amygdala, Etc.)
4.4.1 Receptors
In contrast to the cortical component of the amygdala, the striatal component of
amygdala (e.g., central and medial nuclei) displays much lower levels of CB
1
receptors. CB
1
mRNA in the striatal amygdala was revealed by sensitive in situ
hybridization with the absence of signal in the same region of CB
1
knockout mice
(Marsicano and Lutz 1999).
76 S.S.-J. Hu and K. Mackie
5 Cerebellum and Associated Nuclei
5.1 Cerebellar Cortex
5.1.1 Receptors
The patterns of CB
1
receptor expression in the cerebellum are striking. While
autoradiographic and immunocytochemical studies showed intense labeling of
CB
1
protein in the molecular layer, in situ hybridization studies yielded robust
CB
1
mRNA in the granule cell layer (Matsuda et al. 1990; Herkenham et al. 1991;
Glass et al. 1997; Tsou et al. 1998a; Egertova and Elphick 2000). Purkinje neurons
are devoid of CB
1
protein labeling, while the axon terminals of basket cells
surrounding the Purkinje cell axon initial segment display extremely strong CB
1
labeling. Putting this together, CB
1
receptors are mainly expressed in the axon
terminals of the climbing fibers, parallel fibers, and (some) basket cells, suggesting
a prominent presynaptic localization of CB
1
receptors, mediating modulatory
effects of (endo)cannabinoids at glutamatergic and GABAergic inputs onto
Purkinje neurons. Consistent with this pattern of expression, several elegant
electrophysiological studies demonstrated a role for endocannabinoid inhibition
of glutamatergic and GABAergic neurotransmission onto Purkinje neurons
(Kreitzer and Regehr 2001; Maejima et al. 2001; Diana et al. 2002; Brenowitz
and Regehr 2003). However, additional electrophysiological studies supported the
functional somatic expression of CB
1
receptors (Kreitzer et al. 2002).
5.1.2 Synthetic Enzymes
NAPE-PLD mRNA has been found in the granule cell layer and Purkinje cells, but
not in the molecular layer or white matter (Egertova et al. 2008). However, NAPE-
PLD immunoreactivity is localized in the pre- and post-synaptic areas of the
Purkinje neurons and in the somata of the basket cells in the molecular layer
(Cristino et al. 2008; Suarez et al. 2008).
DAGLαimmunoreactivity is highly concentrated at the base of the spine neck of
cerebellar Purkinje cells. In contrast to its distribution in hippocampal pyramidal
cells, DAGLαis excluded from the main body of spine neck and head (Yoshida
et al. 2006). However, there are no DAGLα-immunoreactive neurons in the granu-
lar layer and in any subdivisions of the inferior olive, suggesting that DAGLαis not
present in parallel and climbing fibers (Suarez et al. 2008).
High levels of DAGLβmRNA have been found in the mouse cerebellar granular
layer by in situ hybridization (Yoshida et al. 2006). However, DAGLβ
immunostaining in the cerebellar cortex is less intense than that of DAGLα.
DAGLβprotein is localized to cell bodies of Purkinje neurons and in the molecular
layer. In contrast to DAGLα, DAGLβ-containing neuropil of the molecular layer
probably represents parallel and climbing fibers from the granular cells and inferior
olive neurons, respectively (Suarez et al. 2008).
Distribution of the Endocannabinoid System in the Central Nervous System 77
5.1.3 Degradative Enzymes
FAAH immunoreactivity is present in the somata and dendrites of the Purkinje
cells, which are innervated by CB
1
-positive axon terminals (Egertova et al. 1998,
2003; Tsou et al. 1998b; Gulyas et al. 2004). Weak FAAH immunostaining is also
evident in the somata of granule cells (Egertova et al. 2003), which is consistent
with the detection of FAAH mRNA in rat cerebellar granule cells (Thomas
et al. 1997).
Both Northern blot and in situ hybridization analyses revealed that MAGL
mRNA is present in rat cerebellum (Dinh et al. 2002), whereas MAGL immunore-
activity is localized to the axon terminals in the molecular layer but absent in the
FAAH-positive Purkinje cell dendrites in rat cerebellum (Gulyas et al. 2004).
Interestingly, a recent immunocytochemical study found that MAGL is heteroge-
neously expressed in mouse cerebellum, with highest levels in parallel fiber
terminals, weak levels in Bergman glia, and complete absence in other synaptic
terminals (Tanimura et al. 2012). Even though the expression of MAGL is limited
to a subset of nerve terminals and astrocytes in the cerebellum, MAGL still
regulates 2-AG retrograde signaling broadly at parallel fiber or climbing fiber to
Purkinje cell synapses (Zhong et al. 2011; Tanimura et al. 2012).
5.2 Deep Cerebellar Nuclei (Fastigial, Interpos, Dentate Nucleus)
5.2.1 Receptors
All cerebellar nuclei (medial, lateral, and interposed nuclei) contain very weak
CB
1
-immunoreactivity throughout the neuropil. However, intense CB
1
immunostaining was found in the neuropil of the dorsal part of the principal nucleus
of the inferior olive (Suarez et al. 2008).
5.2.2 Synthetic Enzymes
Most cerebellar nuclei showed intense neuropil NAPE-PLD immunoreactivity and
a number of moderately NAPE-PLD-labeled neurons. On the other hand, both the
posterior parvicellular part of the interposed cerebellar and lateral cerebellar nuclei
have considerably fewer NAPE-PLD-labelled neurons and a less intense neuropil
immunoreactivity (Suarez et al. 2008).
DAGLαimmunoreactivity is absent in cell bodies of cerebellar and vestibular
nuclei and in other regions with mossy fiber projections in the granular layer such as
the pontine nuclei or the spinal cord (Suarez et al. 2008). In contrast, DAGLβ
immunoreactivity is associated mainly with cell bodies embedded in a network of
fibers. As with NAPE-PLD immunostaining, the posterior parvicellular parts of the
interposed cerebellar and lateral cerebellar nuclei contain fewer DAGLβ-positive
neurons when compared with the remaining cerebellar nuclei (Suarez et al. 2008).
5.2.3 Degradative Enzymes
The strong FAAH immunoreactivity observed in all cerebellar nuclei is related
mainly to the presence of a dense meshwork of fibers, consisting of FAAH-positive
78 S.S.-J. Hu and K. Mackie
punctate labeling that contain immunoreactive somata (Egertova et al. 2003; Suarez
et al. 2008). These cerebellar nuclei are devoid of CB
1
immunoreactivity and
receive synaptic input mainly from Purkinje cell axons. In white matter surrounding
the cerebellar nuclei, FAAH-immunoreactive oligodendrocytes are conspicuous
(Egertova et al. 2003).
Cerebellar and functionally related vestibular nuclei have numerous MAGL-
immunoreactive neurons, showing a perikaryal and dendritic Golgi-like labeling,
similar to that of DAGLβ(Suarez et al. 2008).
6 Brainstem
6.1 Diencephalon
6.1.1 Thalamus (All Nuclei, Including Reticular Thalamic Nucleus,
Habenula)
Receptors
CB
1
receptor expression is very low in most areas of the thalamus, with the
exception of strong labeling in the lateral habenular nucleus, the anterior dorsal
thalamic nucleus, and the reticular thalamic nucleus (Herkenham et al. 1991;
Mailleux and Vanderhaeghen 1992; Tsou et al. 1998a; Marsicano and Lutz
1999). Since CB
1
mRNA is quite abundant in the lateral habenular nucleus,
which has extremely diverse projections, it is likely that (endo)cannabinoids, acting
through CB
1
receptors, significantly affect the diverse functions of the lateral
habenula (Herkenham and Nauta 1977; Herkenham et al. 1991).
Synthetic Enzymes
NAPE-PLD mRNA is found in several thalamic nuclei such as lateral posterior
nuclei and the medial geniculate nucleus, albeit at quite low intensity in mice
(Egertova et al. 2008; Nyilas et al. 2008). In contrast, the highest levels of
NAPE-PLD activity, protein and mRNA, were identified by enzyme assay, western
blotting, and qPCR in rat thalamus, among nine different brain regions examined
(Morishita et al. 2005). In addition, in situ hybridization revealed that moderate
levels of DAGLαmRNA are expressed in mouse thalamus (Yoshida et al. 2006).
Degradative Enzymes
FAAH immunoreactivity has been detected in neuronal somata in the majority of
thalamic nuclei, including the anterodorsal, the anteroventral, the anteromedial, the
ventroanterior, the paratenial, the mediodorsal, the reticulothalamic, the ventrolat-
eral, the ventroposterior, the ventromedial, the posterior, the lateral geniculate, and
the medial geniculate (Egertova et al. 2000,2003). Interestingly, FAAH protein is
much more abundant in these nuclei than the cannabinoid CB
1
receptor, suggesting
its role may be to degrade acyl amides other than anandamide in these regions.
Distribution of the Endocannabinoid System in the Central Nervous System 79
An in situ hybridization study revealed that MAGL mRNA is abundantly
expressed in the anterior thalamus, particularly in the anterodorsal nucleus, whereas
it is sparse in other thalamic nuclei (Dinh et al. 2002).
6.1.2 Hypothalamus (All Nuclei)
Receptors
High levels of CB
1
immunoreactivity have been found in the arcuate,
paraventricular, ventromedial, dorsomedial nuclei, and the external zone of the
median eminence (Wittmann et al. 2007), as well as in the infundibular stem and
lateral hypothalamic area (Tsou et al. 1998a). Further analysis revealed that CB
1
immunoreactivity is detectable in the preterminals of approximately equal numbers
of symmetric and asymmetric synapses, suggesting the occurrence of retrograde
signaling by endocannabinoids in both excitatory and inhibitory hypothalamic
neuronal networks (Wittmann et al. 2007). An in situ hybridization study suggests
that CB
1
mRNA is primarily present on glutamatergic neurons in the hypothalamus
(Marsicano and Lutz 1999). Despite the relatively low levels of CB
1
receptors in the
hypothalamus, functional GTPγS assays revealed these receptors are more effi-
ciently coupled to G proteins than in many other regions (Breivogel and Childers
1998). Finally, a recent study showed that mice with viral-mediated knockdown of
the CB
1
receptor gene (~60 % decrease of the mRNA level) in the hypothalamus,
while maintained on a normocaloric standard diet, displayed decreased body weight
gain over time, subsequent to increased energy expenditure and elevated β
3
-adren-
ergic receptor expression in brown adipose tissues (Cardinal et al. 2012). This result
suggests that hypothalamic CB
1
receptor signaling plays an important role in
energy expenditure under basal conditions, contributing to the antiobesity effect
of CB
1
receptor antagonism.
Synthetic Enzymes
The staining of NAPE-PLD mRNA is evident in cells of the ventromedial nucleus
(Egertova et al. 2008).
6.1.3 Mesencephalon (Colliculi, VTA, PAG, SN, Raphe)
Receptors
A. Substantia Nigra
Both autoradiographic and immunocytochemical studies showed extremely high
levels of CB
1
receptor protein in the substantia nigra (SN) pars reticulata
(Herkenham et al. 1991; Egertova and Elphick 2000). In contrast, in situ
hybridization studies showed very low amounts of CB
1
mRNA in the SN (Matsuda
et al. 1993), suggesting the high levels of CB
1
protein are restricted to incoming
axonal projections from other brain regions. For example, CB
1
receptor protein is
restricted to the GABAergic axonal terminals from the putamen MSNs and the
glutamatergic terminals from the subthalamic nucleus, which may be involved in
80 S.S.-J. Hu and K. Mackie
the control of locomotility by CB
1
activation in the SN (Mailleux and
Vanderhaeghen 1992; Sanudo-Pena and Walker 1997; Sanudo-Pena et al. 1999a).
On the other hand, very low levels of CB
1
receptors exist in sparse intrinsic nigral
neurons, which may exert a direct control on dopaminergic transmission (Matsuda
et al. 1993; Julian et al. 2003). Finally, in rat striatal nerve terminals, a low but
significant percentage of CB
1
–immunoreactivity is co-localized with tyrosine
hydroxylase (TH), a marker for both noradrenergic and dopaminergic terminals
(Kofalvi et al. 2005).
B. Ventral Tegmental Area
Both cannabinoids and endocannabinoids modulate the primary rewarding effects
of many abused drugs, including exogenous cannabinoids, via regulation of drug-
induced increases in dopaminergic neural activity in the VTA (Maldonado
et al. 2006). Therefore, it is of interest to elucidate the expression and function of
CB
1
receptors in the VTA. A sensitive CB
1
polyclonal antibody (Fukudome
et al. 2004) revealed a dense neuropil labeling of CB
1
receptors in the VTA (Matyas
et al. 2008). The CB
1
immunoreactivity is restricted to presynaptic axon terminals
of symmetric synapses, which may belong to local intrinsic GABAergic neurons
(Matyas et al. 2008). Moreover, CB
1
-immunoreactivity is co-localized with vesic-
ular glutamate transporter in presynaptic terminals near dopamine neuron dendrites
in the VAT, indicating the presence of CB
1
receptors in glutamatergic terminals
(Kortleven et al. 2011). Interestingly, co-localization of CB
1
receptor and TH has
been revealed in several brain areas including VTA, thereby pointing to a possible
direct influence of CB
1
receptor activation on dopaminergic neurons (Wenger
et al. 2003). However, further studies are required to clarify this possibility.
C. Periaqueductal Gray
Low to moderate levels of CB
1
receptors have been found in the midbrain
periaqueductal gray (PAG), where the ECS is involved in the control of pain
sensation, including stress-induced analgesia (Walker et al. 1999; Hohmann
et al. 2005; Gregg et al. 2012). In contrast to opiate receptors on GABAergic
aqueductal neurons, CB
1
receptors are preferentially, but not exclusively, localized
in the dorsal portion of the PAG (Tsou et al. 1998a; Azad et al. 2001). In addition,
moderate levels of CB
1
mRNA and protein have been found in the reticular
formation and raphe nucleus, the latter being the main neuronal source of serotonin
in the brain (Glass et al. 1997; Haring et al. 2007), which might have functional
implications in emotion/mood modulation.
Synthetic Enzymes
A. Ventral Tegmental Area
Moderate to high levels of DAGLαare expressed in most neurons of the VTA
(Matyas et al. 2008). High-resolution electron microscopy further revealed that
DAGLαis accumulated in postsynaptic plasma membrane of glutamatergic and
GABAergic synapses, of both TH-positive and negative dendrites. The finding that
Distribution of the Endocannabinoid System in the Central Nervous System 81
DAGLαis present in postsynaptic dendrites juxtaposed to presynaptic CB
1
receptors suggests that 2-AG-CB
1
-mediated retrograde synaptic signaling may
modulate the drug-reward circuitry at multiple types of synapses in the VTA.
B. Periaqueductal Gray
DAGLαprotein is co-localized with mGluR5 within the same dendritic spine heads
at postsynaptic excitatory synapses in rat dorsolateral PAG, which is involved in
2-AG-mediated stress-induced analgesia (Gregg et al. 2012).
Degradative Enzymes
A. Substantia Nigra
FAAH-immunoreactive neurons are not evident in the pars reticulata of the SN,
which contains a very high concentration of CB
1
immunoreactivity in both mouse
and rat (Egertova et al. 2000,2003).
B. Other Nuclei
FAAH-immunoreactive neurons are present in the superior and inferior colliculus,
the rhabdoid nucleus, and several mesencephalic raphe nuclei and are also intensely
stained in the mesencephalic trigeminal nucleus (Egertova et al. 2003). FAAH-
immunoreactive oligodendrocytes associated with fiber tracts are abundant in the
midbrain. For example, FAAH immunostaining was found to be localized in the
myelin sheath surrounding the unstained axons of the mesencephalic trigeminal
tract (Egertova et al. 2003).
6.2 Hindbrain
6.2.1 Medulla (Area Postrema, Cochlear Nuclei, Nucleus
of the Solitary Tract, Trigeminal Nuclei, Various Other Cranial
Nerve Nuclei)
Receptors
Expression of CB
1
receptors, in contrast to the opioid receptors, in the medullary
respiratory control centers is relatively low (Herkenham et al. 1991; Glass
et al. 1997). This likely explains the low mortality caused by cannabinoid intoxica-
tion in humans and animals. However, relatively high levels of CB
1
receptors are
present in the medullary nuclei associated with emesis and vagal control of gut
motility, which may underlie the inhibition of emesis and gastrointestinal motility
by cannabinoids (Krowicki et al. 1999; Van Sickle et al. 2001,2003). For example,
high to moderate levels of CB
1
receptors were found in the area postrema, the
dorsal motor nucleus of the vagus, as well as the medial subnucleus and the
subnucleus gelatinosus of the nucleus of the solitary tract (Van Sickle et al. 2001,
2003; Mackie 2005; Storr and Sharkey 2007).
82 S.S.-J. Hu and K. Mackie
In mouse dorsal cochlear nucleus, CB
1
receptors are highly expressed in
glutamatergic terminals of the parallel fibers, at intermediate levels in glycinergic
terminals, and completely absent in the auditory nerve inputs innervating to the
same DCN principle neurons—fusiform and cartwheel cells (Zhao et al. 2009).
Therefore, CB
1
receptors are well positioned to mediate short- and long-term
plasticities exhibited at parallel fiber synapses, but not at auditory nerve inputs
(Zhao et al. 2011; Zhao and Tzounopoulos 2011).
Synthetic Enzymes
Both DAGLαand DAGLβproteins were identified in the somata of fusiform and
cartwheel cells of mouse DCN, while they were present only in the dendritic spines
of cartwheel cells (Zhao et al. 2009). These findings suggest that the synthesis of
2-AG is more distant from parallel fiber synapses in fusiform than cartwheel cells.
7 Spinal Cord (Dorsal, Ventral, Dorsal Root Ganglion)
7.1 Receptors
Intrathecal application of cannabinoids has been found to suppress pain in various
pain models (Smith and Martin 1992; Welch et al. 1995), which is consistent with
their suppression of noxious stimulus-evoked neuronal firing (Hohmann and
Herkenham 1998) and Fos protein expression in the spinal dorsal horn (Hohmann
et al. 1999b). Moreover, cannabinoids inhibited glutamate release from afferents in
lamina I of dorsal horn in a CB
1
receptor-dependent fashion (Jennings et al. 2001;
Morisset and Urban 2001). Compatible with these functional studies, moderate
levels of CB
1
receptors were found in the superficial layers of the dorsal horn, the
dorsolateral funiculus, and lamina X, all regions of the spinal cord associated with
analgesia (Farquhar-Smith et al. 2000; Nyilas et al. 2009).
However, only a small percentage of CB
1
receptors are localized at central
terminals of primary afferent C fibers, with many more present on large, myelinated
Aβand Aδfibers, as well as postsynaptic interneurons (Hohmann and Herkenham
1998,1999; Hohmann et al. 1999a; Farquhar-Smith et al. 2000). A dorsal rhizot-
omy produced time-dependent 50 % losses in cannabinoid binding densities in the
dorsal horn since rhizotomy destroyed the terminals of both small- and large-
diameter fibers (Hohmann et al. 1999a). However, a quantitative autoradiographic
study showed a modest (16 %) decrease in cannabinoid binding sites in the superfi-
cial dorsal horn by neonatal capsaicin-mediated destruction of sensory C fibers
(Hohmann and Herkenham 1998). Moreover, another study showed little decrease
in CB
1
receptor immunoreactivity following dorsal rhizotomy or hemisection of the
spinal cord, suggesting CB
1
receptors are primarily expressed on interneurons
(Farquhar-Smith et al. 2000). Similarly, CB
1
receptors are only minimally
co-localized with markers for C primary afferents both in the superficial dorsal
horn and dorsal root ganglion (DRG) (Farquhar-Smith et al. 2000; Bridges
et al. 2003). The above data suggest that the majority of CB
1
receptors are not
Distribution of the Endocannabinoid System in the Central Nervous System 83
localized at the presynaptic terminals of nociceptive primary afferents, but rather
may exist on postsynaptic interneurons (Farquhar-Smith et al. 2000; Salio
et al. 2002), a CB
1
distribution which differs from the strong presynaptic axon
terminal localization of CB
1
receptors in most other brain regions (Katona
et al. 1999; Nyiri et al. 2005a). Indeed, research showed that CB
1
receptors
localized at dorsal horn inhibitory postsynaptic interneurons mediate C-fiber-
induced pain sensitization. However, it is important to emphasize the unexpected
pro-nociceptive role of endocannabinoids is specific for C-fiber-mediated activity-
dependent hyperalgesia, in contrast to the anti-nociceptive effect of these endoge-
nous lipid molecules in models of inflammatory and neuropathic pain (Pernia-
Andrade et al. 2009). Moreover, a conditional nociceptor-specific loss of CB
1
was found to reduce spinal CB
1
-specific ligand binding by approximately 20 %
only and did not substantially decrease CB
1
-immunoreactivity in spinal laminae I
and II (Agarwal et al. 2007). However, this conditional knockout of CB
1
in
peripheral nociceptive neurons led to a substantial reduction of analgesia produced
by local and systemic delivery of cannabinoids, suggesting that low levels of CB
1
expression did not necessarily mean lack of functional significance (Agarwal
et al. 2007). Therefore, it is likely that the interplay between cannabinoid actions
on peripheral primary afferents, interneurons, and descending pathways collec-
tively contributes to the analgesic effects of CB
1
receptor activation in the
spinal cord.
Interestingly, despite the earlier reports of minimal localization of CB
1
receptors
in nociceptive DRG neurons (Hohmann and Herkenham 1999; Bridges et al. 2003),
more recent studies suggest a much broader (40–80 %) distribution of CB
1
receptors in nociceptive neurons of DRG and trigeminal ganglia (Mitrirattanakul
et al. 2006; Agarwal et al. 2007). While the possible reasons for these discrepancies
have been well discussed elsewhere (Marsicano and Kuner 2008), it is also impor-
tant to note that the expression and peripheral transport of CB
1
receptors in the
DRG can be upregulated by peripheral inflammation (Amaya et al. 2006). Finally,
some immunocytochemical evidence suggests CB
1
receptors are also present in the
ventral horn (Tsou et al. 1998a; Sanudo-Pena et al. 1999b), a spinal cord area
associated with movement.
7.2 Synthetic Enzymes
DAGLαmRNA is widely expressed in spinal dorsal horn neurons (Nyilas
et al. 2009). Similar to CB
1
receptors, high levels DAGLαprotein have been
found to be localized at the superficial dorsal horn. High-resolution electron
microscopy demonstrated a postsynaptic localization of DAGLαat nociceptive
synapses, which is juxtaposed to the presynaptic CB
1
-containing excitatory primary
afferents (Nyilas et al. 2009) (Fig. 1B, C). Interestingly, postsynaptic DAGLαis
co-localized with mGluR5, whose activation induces 2-AG biosynthesis (Nyilas
et al. 2009).
84 S.S.-J. Hu and K. Mackie
7.3 Degradative Enzymes
FAAH has been found in the cell bodies of ventral horn neurons (Tsou et al. 1998b).
The presence of FAAH and CB
1
receptors in circuits involved in spinal reflexes
may underlie the antispastic effects of (endo)cannabinoids.
8 Summary, Concluding Thoughts, and Future Directions
As this brief review has shown, the components of the endocannabinoid system
(ECS) are widespread throughout the CNS. Endocannabinoids have a major role as
retrograde transmitters in many brain regions, although often with region-specific
specialization as discussed above. Thus, in most brain regions, the highest levels of
CB
1
receptors are found presynaptically, while endocannabinoid synthesizing
enzymes are present postsynaptically. Interestingly, some endocannabinoid
degrading enzymes are found presynaptically (e.g., MAGL) and others postsynap-
tically (e.g., ABHD6 and FAAH), suggesting specialized roles of each in
endocannabinoid degradation (Gulyas et al. 2004; Blankman et al. 2007), and
introducing an extra layer of complexity in endocannabinoid metabolism. A varia-
tion on the presynaptic localization of CB
1
is the somatic expression of CB
1
, most
commonly in some cortical and cerebellar neurons, where endocannabinoid signal-
ing is cell autonomous. Future studies are needed to better define the distribution of
some less-well studied (putative) ECS components, including α/β-hydrolase
domain 4 (ABHD4) (Simon and Cravatt 2006), glycerophosphodiesterase
1 (GDE1) (Simon and Cravatt 2006,2010), ABHD6 (Blankman et al. 2007;
Marrs et al. 2010), α/β-hydrolase domain 12 (ABHD12) (Blankman et al. 2007),
cannabinoid receptor interacting protein 1a (CRIP1a) (Niehaus et al. 2007), and
DAGLβ(Gao et al. 2010).
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