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RESEARCH ARTICLE
Spatial distribution of cannabinoid receptor
type 1 (CB
1
) in normal canine central and
peripheral nervous system
Jessica Freundt-Revilla
1,2☯
*, Kristel Kegler
2,3☯¤
, Wolfgang Baumga
¨rtner
2,3
,
Andrea Tipold
1,2
1Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover
Foundation, Hannover, Germany, 2Center for Systems Neuroscience, Hannover, Germany, 3Department
of Pathology, University of Veterinary Medicine Hannover Foundation, Hannover, Germany
☯These authors contributed equally to this work.
¤Current address: Institute for Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland
*Jessica.Freundt.Revilla@tiho-hannover.de
Abstract
The endocannabinoid system is a regulatory pathway consisting of two main types of canna-
binoid receptors (CB
1
and CB
2
) and their endogenous ligands, the endocannabinoids. The
CB
1
receptor is highly expressed in the central and peripheral nervous systems (PNS) in
mammalians and is involved in neuromodulatory functions. Since endocannabinoids were
shown to be elevated in cerebrospinal fluid of epileptic dogs, knowledge about the species
specific CB receptor expression in the nervous system is required. Therefore, we assessed
the spatial distribution of CB
1
receptors in the normal canine CNS and PNS. Immunohis-
tochemistry of several regions of the brain, spinal cord and peripheral nerves from a healthy
four-week-old puppy, three six-month-old dogs, and one ten-year-old dog revealed strong
dot-like immunoreactivity in the neuropil of the cerebral cortex, Cornu Ammonis (CA) and
dentate gyrus of the hippocampus, midbrain, cerebellum, medulla oblongata and grey mat-
ter of the spinal cord. Dense CB
1
expression was found in fibres of the globus pallidus and
substantia nigra surrounding immunonegative neurons. Astrocytes were constantly positive
in all examined regions. CB
1
labelled neurons and satellite cells of the dorsal root ganglia,
and myelinating Schwann cells in the PNS. These results demonstrate for the first time the
spatial distribution of CB
1
receptors in the healthy canine CNS and PNS. These results can
be used as a basis for further studies aiming to elucidate the physiological consequences of
this particular anatomical and cellular distribution.
Introduction
The properties for medical intervention of the plant Marijuana (Cannabis sativa) have been
known for centuries [1,2]. Behavioural and pharmacological effects of its most psychoactive
component, Δ
9
–tetrahydrocannabinol (THC), can be explained by the activation of receptors
localized in the nervous system [3,4] and peripheral tissues [5]. These receptors are known as
PLOS ONE | https://doi.org/10.1371/journal.pone.0181064 July 10, 2017 1 / 21
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OPEN ACCESS
Citation: Freundt-Revilla J, Kegler K, Baumga¨rtner
W, Tipold A (2017) Spatial distribution of
cannabinoid receptor type 1 (CB
1
) in normal canine
central and peripheral nervous system. PLoS ONE
12(7): e0181064. https://doi.org/10.1371/journal.
pone.0181064
Editor: Faramarz Dehghani, Martin Luther
University, GERMANY
Received: November 18, 2016
Accepted: June 26, 2017
Published: July 10, 2017
Copyright: ©2017 Freundt-Revilla et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data is
contained within the paper.
Funding: JFR recieved funding from the Deutscher
Akademischer Austauschdienst (DAAD, Germany),
Grant number: GR 6220 22681, https://www.daad.
de. The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
cannabinoid receptors (CBs), and along with their endogenous ligands, the endocannabinoids
(ECs), and the enzymes responsible for their synthesis and degradation, constitute the endo-
cannabinoid system [6,7]. In mammalian tissues, two main subtypes of cannabinoid recep-
tors, the cannabinoid receptor 1 (CB
1
) and cannabinoid receptor 2 (CB
2
), which are G
protein-coupled receptors, have been recognized [5,8] and are responsible for the transduc-
tion of different effects of ECs [9]. Furthermore, CB
1
receptors have been shown to be primar-
ily expressed in the central nervous system (CNS) and peripheral nervous system (PNS) while
CB
2
receptors are mostly found in cells of the immune system [10,11].
Besides the therapeutical effects of several cannabinoids as antiemetics, analgesics, antispas-
modics, appetite-stimulating, and sleeping inductors [12]; THC, a phytocannabinoid partial
CB
1
agonist, as well as WIN55,212–2, a synthetic CB
1
agonist, have both been proved to have
an anticonvulsant effect in vitro [13] and in rodent models [14] of epilepsy and status epilepti-
cus, being more effective than conventional antiepileptics like phenytoin and phenobarbital
[15]. Furthermore, increased levels of anandamide (AEA), an endocannabinoid, have been
found in cerebrospinal fluid of dogs suffering from idiopathic epilepsy compared to healthy
dogs [16]. Several companies started to sell medical marihuana to be used in pets to treat
chronic pain, seizures, inflammation, cancer, diabetes, nausea, anxiety and obesity. There is,
however, a lack of reliable research to back those claims regarding the specific distribution of
cannabinoid receptors and their associated function according to their presence in different
anatomical localization within the healthy nervous system and under pathological conditions.
The expression of CB
1
has been described in brain sections of humans using autoradiogra-
phy [3,17] and in rhesus monkeys by positron emission tomography (PET) [18]. Immunohis-
tochemistry allows the identification of particular neuronal cells and fibres that express
cannabinoid receptors because of its greater resolution [19]. Consequently, CB
1
distribution
has been extensively mapped in the mouse [20], rat [19,21] and macaque monkey [22] CNS.
In addition, CB
1
expression has also been described particularly in the dorsal horn in rats [19,
23] and in the spinal cord of humans [17]. In the species dog, CB
1
receptors were detected in
salivary glands [24], hair follicles [25], skin and hippocampus [26]. However, a detailed analy-
sis of the distribution of CB
1
receptors in the CNS and PNS has not been reported in canines
so far. It is well established that many conditions in dogs share striking similarities with their
human counterparts thus representing suitable translational models for studying human neu-
rological diseases including epilepsy [27], neuropathic pain [28], spinal cord injury [29] and
multiple sclerosis, as described in the canine distemper virus (CDV)-induced demyelination
model [30]. Thus, the species dog might help to overcome the gap between highly homogenous
and standardized rodent models and clinically relevant conditions in humans. Precise knowl-
edge of the distribution of CB
1
within the canine nervous system are therefore of great rele-
vance to design therapeutic strategies to manipulate the effects of the endocannabinoid system
[9].
In the current study, we analyzed the spatial distribution of CB
1
receptors in the healthy
CNS and PNS of dogs from different ages. This is the first study which characterizes in detail
the presence of those receptors under normal circumstances, therewith providing novel
insights into the localization of CB
1
receptors for further characterization under pathophysio-
logical conditions.
Materials and methods
Animals and tissue samples
Following routine necropsy, brain and peripheral nerve samples of dogs without clinical or
pathological evidence of neurologic or infectious diseases were collected and subsequently
Cannabinoid receptor type 1 in canine nervous system
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fixed in non-buffered formalin (10%) for at least 48 hours and embedded in paraffin. Serial
sections (3 μm thick) were mounted on SuperFrost-Plus slides (Menzel Gla¨ser, Braunschweig,
Germany), and stained with hematoxylin and eosin (HE), a complete histological examination
was performed in order to confirm the absence of histopathological lesions. Afterwards, the
slides where further processed for immunohistochemistry and double immunofluorescence. A
total of five dogs of different ages were included, one female and two male six-month-old Bea-
gle dogs, one ten-year-old female Cocker Spaniel and one four-week-old female Leonberger.
Tissue samples of the dogs used in this study were included in a previous study [31]. German
Animal Welfare Act with the law of animal welfare, Germany (permission number: 33.9-
42502-05-13A346), and the ethical guidelines of the University of Veterinary Medicine Han-
nover were followed for the euthanasia of the dogs. No animals were euthanized for this partic-
ular study; samples obtained and previously used in other studies were taken. The study was
approved and followed the guidelines of the PhD commission of the University of Veterinary
Medicine Hannover, the institutional ethics committee.
Transversal sections were cut through the brain at the level of olfactory bulb, frontal lobes,
basal forebrain, thalamus, lateral and medial corpus geniculatum, hippocampus, cerebellum
and brainstem. Transversal sections of the cervical, thoracic and lumbar spinal cord with their
corresponding dorsal root ganglia were included, as well as a representative section of the sci-
atic nerve.
Antibodies
For immunohistochemistry (IHC) and immunofluorescence (IF) a polyclonal antibody against
cannabinoid receptor 1 (CB
1
, Abcam Cat# ab23703, RRID:AB_447623, 1:100 IHC, 1:15 IF),
immunogen corresponding to C terminal amino acids 461–472 of Human Cannabinoid recep-
tor 1, was included. Monoclonal antibodies included anti-glial fibrillary acidic protein (GFAP,
Sigma-Aldrich Cat# G-A-5, RRID:AB_2314539, 1:300 IF), anti-2’,3’-Cyclic-nucleotide 3’-
phosphodiesterase (CNPase, Millipore Cat# MAB326, RRID:AB_2082608, 1:100 IF), anti-
major peripheral myelin protein (P0, clone P07, 1:400 IF, Archelos et al., 1993) and anti-neu-
rotrophin receptor p75 (p75
NTR
, American Type Culture Collection (ATCC) Cat# hb-8737,
RRID:AB_2152662, 1:2 IF).
Immunohistochemistry
CB
1
immunohistochemistry (IHC) was performed by using the avidin-biotin-peroxidase com-
plex (ABC) method as previously described [31,32]. Briefly, 3 μm thick sections were dewaxed
and rehydrated through a graded series of alcohols, and treated with 0.5% H
2
O
2
to block endog-
enous peroxidase. Antigenic retrieval was preformed using sodium-citrate buffer (pH 6.0–6.5)
for 20 minutes in the microwave at 800w. Following incubation with 20% goat serum, sections
were incubated with the CB
1
antibody overnight at 4˚C. As negative control, the primary anti-
body was substituted with rabbit serum (1:3000; R4505; Sigma Aldrich, Taufkirchen, Germany),
using the same gamma-globulin concentration as in the primary antibody formulation. Biotiny-
lated goat-anti-rabbit IgG (1:200; BA-1000; Vector Laboratories, Burlingame, CA, USA), was
used as secondary antibody and incubated for 45 minutes at room temperature, followed by
incubation with ABC (VECTASTAIN-ABC Kit Standard, PK 6100, Vector Laboratories, Bur-
linghame, California, USA). Color development was done with 3.3’-diaminobenzidine tetrahy-
drochloride (0.05% solution, DAB, Sigma Aldrich, Taufkirchen, Germany) with H
2
O
2
(0.03%,
pH 7.2) for 5 min followed by slight counterstaining with Mayer’s hemalaun. Sections of tissue
samples were independently examined via light microscopy (BX51, Olympus Optical CO.,
Cannabinoid receptor type 1 in canine nervous system
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Tokyo, Japan). Representative images were acquired by use of photodocumentation software
(DP72, Olympus Optical CO., Tokyo, Japan).
Double immunofluorescence staining
Double immunofluorescence staining was performed on representative tissue sections as pre-
viously described [32] on 3 μm thick paraffin-embedded sections to demonstrate a possible
co-localization of CB
1
with GFAP and CNPase in the CNS, and P0 and p75
NTR
in the PNS.
Briefly, sections were simultaneously incubated with the respective primary antibodies for 90
min. Cy3-labeled goat anti-mouse (red, 1:200, Alexa Fluor 555 dye, Life Technologies) and
Cy2-labeled goat anti-rabbit (green, 1:200, Alexa Fluor 488 dye, Life Technologies) secondary
antibodies were used to visualize the respective antigens. Nuclear counterstaining was per-
formed with 0.01% bisbenzimide (H33258, Sigma Aldrich, Taufkirchen, Germany) and sec-
tions were mounted with Dako Fluorescent Mounting medium (DakoCytomation, Hamburg,
Germany). Antigenic expression was visualized using an inverted fluorescence microscope
(BZ-9000E, Keyence GmbH, Neu-Isenburg, Germany) and examined through the BZ-II Ana-
lyzer software. All images were acquired with the same microscope settings under which con-
trol sections showed no signal. Images were transferred to Adobe Photoshop (San Jose, CA)
for cropping, and they were adjusted to optimize contrast and brightness.
Results
The distribution of CB
1
immunoreactivity in anatomically related regions is described below
in detail. Importantly, there were few differences in the expression of CB
1
regarding the ana-
lysed anatomical localisations in different aged dogs. Generally, strong cytoplasmic CB
1
immunoreactivity was observed in astrocytes both in the white and in the grey matter along
the cerebrum (Fig 1A), cerebellum and spinal cord in all dogs, except in the four-week-old
dog, in which only scattered astrocytes were slightly positive (Fig 1B). In addition, the cyto-
plasm of ependymal cells lining the lateral (Fig 1C and 1D), third, fourth (Fig 1E) ventricles
and the central canal of the spinal cord; as well the choroid plexus ependymal cells (Fig 1F)
strongly expressed CB
1
. Strikingly, the cytoplasm of small numbers of neuroglial cells sur-
rounding the fourth ventricle (Fig 1E) and the central canal of the spinal cord were intensely
CB
1
positive. Within the meninges, flattened fibroblast-like cells mostly in the dura mater
showed slight cytoplasmic CB
1
immunoreactivity.
Olfactory bulb
In the main olfactory bulb, network of fibres were intensely stained with CB
1
in the glomerular
layer (GL) (Fig 2A). CB
1
immunoreactivity occurred in a network of fibres that surrounded
unstained neuronal soma (Fig 2B). Immunoreactivity was also found in the fibres of the inter-
nal plexiform layer (IPL) (Fig 2A). In addition, a population of cells within the internal granule
cell layer were strongly CB
1
positive (Fig 2C). No immunoreactivity was observed in the exter-
nal plexiform layer (EPL) or in the mitral cell layer (ML). However, mitral cells axons were
moderately CB
1
positive. In the four-week-old dog, only the glomerular layer expressed mod-
erate CB
1
immunoreactivity and all other layers were devoid of immunostaining (Fig 2D).
Cerebral cortex (neocortex-frontal lobe)
The grey matter of the neocortex expressed strong CB
1
immunoreactivity in the external gran-
ular layer (II), external pyramidal layer (III), inner granular layer (IV), inner pyramidal layer
(V) and multiform layer (VI) (Fig 3A and 3B). This intense immunoreactivity of the fibres was
Cannabinoid receptor type 1 in canine nervous system
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Fig 1. CB
1
immunoreactivity. Astrocytes (arrow) of the cerebral white matter of a six-month-old Beagle dog showing strongCB
1
receptor immunoreactivity (A) comparing to astrocytes of a four-week-old dog, which are only slightly positive (B). The ependymal cells
(arrow) of a six-month-old dog lining the lateral ventricle strongly express CB
1
receptor (C, D). Similarly, ependymal cells lining the fourth
ventricle and scattered neuroglial cells (E) are CB
1
receptor positive, as well as cells of the choroid plexus (F). IHC was performed using
the avidin-biotin-peroxidase complex (ABC) method.
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Cannabinoid receptor type 1 in canine nervous system
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presented in a dot-like pattern surrounding the unstained neuronal bodies (Fig 3A). The dens-
est expression was found in the II, III, V and VI layers of the frontal lobe while the molecular
layer (I) appeared almost devoid of CB
1
immunostaining (Fig 3B). In the four-week-old dog
and the ten-year-old dog, the intensity of the immunoreactivity was lower comparing to the
other dogs.
Hippocampus
Within the hippocampus, strong dot-like CB
1
immunostaining was associated with a dense
network of fibres in the stratum pyramidale surrounding the unstained pyramidal neuronal
bodies (Fig 3C and 3E). A progressive decrease in the immunoreactivity from CA1 to CA4 was
seen (Fig 3C). In the hippocampal polymorphic layer and the molecular layer, the fibres were
less intensely stained. In the dentate gyrus, CB
1
immunoreactivity was associated with fibres in
the molecular layer with the most intense staining occurring adjacent to the granule cell layer
(Fig 3D). The granule cell layer lacked CB
1
expression (Fig 3D).
Fig 2. CB
1
immunoreactivity of the Olfactory bulb. CB
1
immunoreactivity of a six-month-old Beagle dog (A, B, C) and four-week-old dog (D).
Strong immunoreactivity of the glomerular layer (GL), lack of immunoreactivity in the external plexiform layer (EPL) and mitral cell layer (ML), while
moderate immunoreactivity of the internal plexiform layer (IPL) are observed in the six-month-old Beagle dog (A). Detailed immunoreactivity of the
GL (arrow) is depicted in B. In the six-month-old Beagle dog, a population of cells within the internal granule cell layer (arrow) is strongly CB
1
receptor positive (C). Contrary, the glomerular layer in the four-week-old dog was only slightly CB
1
receptor positive (D). IHC was performed using
the avidin-biotin-peroxidase complex (ABC) method.
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Cannabinoid receptor type 1 in canine nervous system
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Fig 3. CB
1
immunostaining of the cerebral cortex, hippocampus and substantia nigra in a six-month-old Beagle dog. Within the frontal
lobe of the cerebral cortex, there is an intense CB
1
immunoreactivity of fibres surrounding unstained neuronal bodies in layers V andVI (A;
arrow). In figure B, layer I appears almost devoid of CB
1
immunoreactivity and layer II and III express strong immunoreactivity. Notice that the
meninges show positive flattened fibroblast-like cells in the dura matter (B, arrow). The hippocampus shows progressive decrease in the
immunoreactivity from C1 to C4 (C). In figure D, the dentate gyrus of the hippocampus depicting strong dot-like CB
1
immunoreactivity in the
molecular layer (ML). Interestingly, the granule cell layer (GCL) appears devoid of CB
1
immunoreactivity. The stratum pyramidale shows strong
CB
1
immunoreactive fibres surrounding unstained pyramidal neuronal bodies in the C1 (E; arrow). In figure F, strong CB
1
immunoreactivity is
observed in fibres of the substantia nigra pars reticulata. IHC was performed using the avidin-biotin-peroxidase complex (ABC) method. WM:
white matter; GM: grey matter.
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Cannabinoid receptor type 1 in canine nervous system
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Basal ganglia and lateral and medial geniculate nucleus
Intense CB
1
immunoreactive fibres were observed in the globus pallidus of the basal nuclei.
The expression of CB
1
was observed only in scattered fibres in the lateral and medial geniculate
nucleus.
Midbrain
Strong CB
1
immunoreactivity was observed in the fibres surrounding unstained neuronal bod-
ies in the substantia nigra, denser towards pars reticulata (Fig 3F). There were intensely stained
fibres from all directions at the level of the oculomotor nucleus and red nucleus. In addition,
moderate CB
1
immunoreactivity was observed in the fibres of the periaqueductal gray (PAG)
and in the soma of neurons.
Cerebellum
Strong CB
1
immunoreactivity was observed homogenously within the molecular layer of
the cerebellar cortex (Fig 4A and 4B). Small numbers of Purkinje cells showed slight cyto-
plasmic immunoreactivity. Interestingly, strong immunoreactivity was present surrounding
the Purkinje cells bodies, particularly in the basal portion of the cells (Fig 4B). The underly-
ing granule cell layer remained negative, with just few scattered positive fibres surrounding
unstained cellular bodies (Fig 4B). In the ten-year-old dog the staining pattern remained
alike, however, the molecular layer showed moderate to slight CB
1
immunoreactivity, while
other layers remained negative (Fig 4C and 4D), Purkinje cells were surrounded by dot
immunoreactivity (Fig 4D).
Medulla oblongata
Surrounding the neuronal bodies of the cochlear nucleus (Fig 4E) and the nucleus of the spinal
tract of the trigeminus, a strong dot-like CB
1
immunoreactivity was observed while the neuro-
nal cytoplasm were completely negative (Fig 4F).
Spinal cord
Within the grey matter of the cervical, thoracic and lumbar spinal cord, strong CB
1
immunoreactive fibres were observed in the dorsal horn, intermediate region and ventral
horn (Fig 5A). CB
1
dot-like immunostaining was present surrounding the neuronal bod-
ies (Fig 5B). In addition very few neurons showed slight cytoplasmic immunoreactivity
within the ventral and dorsal horns (Fig 5B). In the four-week-old and the ten-year-old
dogs, the intensity of the immunoreactivity in the grey matter was lower comparing to
the six-month-old dogs (Fig 5C).
Dorsal root ganglia
Within the dorsal root ganglia (DRG) of the cervical, thoracic and lumbar spinal cord seg-
ments large neurons showed slight cytoplasmic CB
1
immunoreactivity, while small dark
neurons strongly expressed CB
1
(Fig 5D). In addition, satellite cells were strongly immu-
nopositive (Fig 5D). In the ten-year-old dog the immunostaining pattern remained, nev-
ertheless, the overall DRG immunostaining was weaker (Fig 5E). In the four-week-old,
however, only scattered large and small dark neurons and satellite cells were slightly posi-
tive (Fig 5F).
Cannabinoid receptor type 1 in canine nervous system
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Fig 4. CB
1
immunoreactivity of the cerebellum and choclear nuclei. In figure A notice strong CB
1
immunoreactivity within the molecular
layer of the cerebellar cortex in a six-month-old Beagle dog. Figure B depicting in detail immunonegative Purkinje cells surrounded by strong
immunorreactive fibers particularly in the basal portion (arrow). In the ten-year-old dog, there is a slight immunoreactivity in the molecular
layer of the cerebellar cortex (C). Purkinje cells surrounded by a dot-like immunoreactivity appear devoid of immunoreactivity in the ten-year-
old dog (D; arrow). The cochlear nucleus in a six-month-old dog showing strongCB
1
immunoreactivity (E). In figure F detail of the cochlear
nucleus with strong CB
1
immunoreactivity surrounding the unstained neuronal bodies (arrow). IHC was performed using the avidin-biotin-
peroxidase complex (ABC) method.
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Peripheral nerve
CB
1
immunostaining within the thoracic spinal nerve revealed strong expression in randomly
distributed Schwann cells ensheathing axons (Fig 5G). In the four-week-old and the ten-year-
old dogs, the intensity of the CB
1
immunoreactivity was lower in positive Schwann cells (Fig
5H and 5I). Moreover, in the ten-year-old dog only few Schwann cells showed a moderate pos-
itive immunoreactivity (Fig 5H).
Fig 5. CB
1
immunoreactivity in the spinal cord, dorsal root ganglia and peripheral nerve. In figure A, strong CB
1
immunoreactivity is shown in the grey
matter of the cervical spinal cord of a six-month-old dog and the cytoplasm of ependymal cells lining the central canal (A; arrow). Within the dorsal horn, CB
1
immunoreactivity appears surrounding unstained neuronal bodies (B; arrow). In the cervical spinal cord of a ten-year-old dog notice slight immunoreactivity of
the grey matter (C). Figure D showing the cervical dorsal root ganglia of a six-month-old dog with slight immunoreactivity of large neurons and strong CB
1
immunoreactivity of small dark neurons (arrows) and satellite cells (arrowheads). The thoracic dorsal root ganglia of a ten-year-old dog with moderate CB
1
immunoreactivity of small dark neurons and satellite cells, large neurons show slight immunoreactivity (E; arrow). The cervical dorsal root ganglia of a four-
week-old dog depicting scattered large and small neurons and satellite cells with slight CB
1
immunoreactivity (F; arrow). In figure G the cervical spinal nerve
of a six-month-old dog shows strong CB
1
expression in Schwann cells ensheating axons (arrow). Few Schwann cells show moderate CB
1
immunoreactivity
(arrow) in a thoracic spinal nerve of a ten-year-old dog (H). The cervical spinal nerve in the four-week-old dog shows moderate CB
1
immunoreactivity of
scattered Schwann cells (I; arrow). IHC was performed using the avidin-biotin-peroxidase complex (ABC) method.
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Double immunofluorescence tracking of glial cells expressing CB
1
receptors in the CNS and PNS
In order to specifically identify the glial cell types expressing CB
1
receptors in the CNS and
PNS, double immunofluorescence was performed in a representative case of a six-month-old
beagle dog. Co-localization of CB
1
with the astrocytic marker GFAP was observed in about
20% of the GFAP-positive astrocytes, indicating that only a subpopulation of astrocytes does
express CB
1
receptors (Fig 6A and 6C). On the other hand, no co-expression was present
among CB
1
and the mature oligodendrocytic marker CNPase (Fig 6D and 6F).
Interestingly, double immunolabelling of the sciatic nerve showed co-localization of CB
1
and P0, a marker for myelinating Schwan cells, in about 100% of the Schwann cells (Fig 7A
and 7C). On the contrary, no co-expression was found among CB
1
and the non-myelinating
Schwann cells marker p75
NTR
(Fig 7D and 7F).
Discussion
This study describes the first detailed spatial distribution of CB
1
receptors in the healthy canine
CNS and PNS. A commercially available antibody against human CB
1
was used, correspond-
ing to C terminal amino acids 461–472 of human cannabinoid receptor type 1. The CB
1
pro-
tein sequence is highly conserved across mammalian species [33], moreover, crossreactivity of
this specific CB
1
antibody with canine tissue has been previously demonstrated in peripheral
tissues, hippocampus and cerebellum of adult dogs [26,34] and in canine embryos [35].
The distribution of CB
1
receptors in the CNS of dogs in this study was similar but no equal
to those of previews studies made in rats [21] and monkeys [22] using C-terminus antibodies,
and those of mice [20], rats [19] and monkeys [22] using N-terminus antibodies.
Fig 6. Double immunofluorescence staining of the cerebral white matter of a six-month-old Beagle dog. Double immunofluorescence staining of
CB
1
(green, A) with GFAP (red, B) reveals co-localization in about 20% astrocytes (C). CNPase expression (red, E) and CB
1
(green, D) do not co-localize,
suggesting a lack of expression of CB
1
receptors by mature oligodendrocytes (F). Nuclear staining (blue) with bisbenzimide.
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Cannabinoid receptor type 1 in canine nervous system
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The presence of cannabinoid receptors in the canine CNS was first reported by the autora-
diographic studies of Herkenham in 1990, who found the densest binding of a radiolabeled
synthetic cannabinoid in the cerebellar molecular layer, followed by the globus pallidus, sub-
stantia nigra pars reticulate, hippocampal dentate gyrus and the neocortex [3]. This pattern,
with few variations remained for humans, rhesus monkeys, rats and guinea pigs [3]. Our study
using immunohistochemistry shows similar results regarding the overall distribution of CB
1
in
healthy CNS of dogs.
In the current study, the distribution of CB
1
immunostaining consisted mostly on strongly
positive network of fibres in specific regions such as the olfactory bulb, cerebral cortex, cere-
bellar cortex, hippocampus, basal ganglia, cochlear nucleus, nucleus of the spinal tract of the
trigeminus and grey matter of the spinal cord. This particular distribution might be due to the
fact that the CB
1
receptors are mainly expressed in axons and pre synaptic terminals [36–38],
emphasizing the important role of this receptor as a modulator of neurotransmitter release at
specific synapses [9,39,40]. CB
1
receptors, however, have also been found on postsynaptic
structures [22,23,41], glial cells [42–45] and peripheral cells such as cells of the striated ducts
of the parotid and mandibular glands, keratinocytes, fibroblasts and macrophages [24,25,46–
48].
Strikingly, ependymal cells lining the ventricular system and the central canal of the spinal
cord, and a small numbers of neuroglial cells surrounding the fourth ventricle and the central
canal expressed the strongest cytoplasmic CB
1
immunoreactivity. Cells surrounding the cen-
tral canal of the spinal cord are a source of stem/precursor cells that may give rise to neurons,
astrocytes, or oligodendrocytes [49]. The ependymal region in the spinal cord has been shown
to express CB
1
in rodents [50] and humans [51]. Even a subpopulation of ependymal cells
Fig 7. Double immunofluorescence staining of the sciatic nerve of a six-month-old dog. P0, a markerfor myelinating Schwann cells (red, B) and CB
1
(green, A) co-localize in about 100% of Schwann cells (C). p75
NTR
(red, E) and CB
1
(green, D) do not co-localize (F), suggesting the absence of CB
1
receptors in non-myelinating Schwann cells. Nuclear staining (blue) with bisbenzimid.
https://doi.org/10.1371/journal.pone.0181064.g007
Cannabinoid receptor type 1 in canine nervous system
PLOS ONE | https://doi.org/10.1371/journal.pone.0181064 July 10, 2017 12 / 21
named “CB
1
high cell” has been described in both species [49,51], co-expressing stem/precur-
sor cell markers in rats [49]. Control of proliferation of brain progenitors/stem cells through
CB receptor activation has been shown in vitro [52,53]. Furthermore, “CB
1
high cells” prolifer-
ate during early postnatal development and after spinal cord injury (SCI) in adult rats, but not
in the unlesioned spinal cord [49]. Aguado and others showed that endocannabinoid signal-
ling controls neural progenitor differentiation into astroglial cells in postnatal and adult mice
[52]. Nevertheless, further studies are needed to fully understand the potential of cannabinoids
on neurogenesis in the dog and other species [49].
In the olfactory bulb, we demonstrated a network of fibres intensely stained with CB
1
in the
glomerular layer and moderate staining of the internal plexiform layer surrounding unstained
neuronal soma. Despite lack of immunostaining of the mitral cell layer, mitral cell axons were
moderately CB
1
positive. Furthermore, a population of cells located in the internal granule cell
layer expressed strong cytoplasmic CB
1
immunoreactivity. In mice [54] and rats [19,21], the
strongest CB
1
immunoreactivity has been detected in the fibres of the inner granule cell layer,
followed by the inner plexiform layer, however, only surrounding unstained cell bodies. Soria-
Go
´mez and others demonstrated that the endocannabinoid system controls food intake via
olfactory processes in mice [54].
CB
1
receptors are described to be densely expressed in all regions of the cortex in mice, rats,
monkeys and humans [17,19,22,55]. While the general laminar pattern of CB
1
immunoreac-
tivity between species seems preserved [9], the densest expression appears to be in the III and
V in primates [9] and within layers II, III and VI in mice [21] and rats [19], while layer I
appears almost devoid of CB
1
receptors in these species [9,21]. We found intense CB
1
immu-
noreactivity presented in a dot-like pattern of the fibres surrounding unstained neuronal bod-
ies, as previously described [21], within layers II, III, IV, V, and VI of the neocortex, with the
densest expression found in the II, III, IV and VI layers of the frontal lobe. In humans and
monkeys, the laminar pattern has been widely studied and shows distinctive laminar density
across the different regions, showing higher CB
1
expression in the prefrontal cortex [22]. In
the monkey, neocortex immunoreactivity is primarily contained in cells and axon terminals
that show morphological features of GABAergic neurons [22]. Moreover, the majority of high
CB
1
expressing cells in the rat forebrain are GABAergic neurons [56].
The strongest immunoreactivity we found in the hippocampus was associated with fibres
within the molecular layer of the dentate gyrus with the most intense staining occurring adja-
cent to the granule cell layer, which lacked CB
1
expression. A similar pattern has been found
in macaques [22], mice [20] and rats [19], where the highest CB
1
density was found within the
molecular layer of the dentate gyrus, while the granule cell layer appeared to be completely
devoid of CB
1
immunoreactivity. In immunohistochemical studies made in rats [19], mice
[20,57] and macaques [22] strong immunoreactivity occurred in the Cornu Ammonis (CA)
regions of the hippocampus within the pyramidal layer. Interestingly, the cell bodies of pyra-
midal neurons in CA1-CA3 fields appeared to be unstained but surrounded by a dense plexus
of highly immunoreactive fibres [19–22,57]. Indeed, we found strong CB
1
immunostaining
associated with a dense network of fibres in the stratum pyramidale also surrounding immu-
nonegative pyramidal neuronal bodies. Specific expression of CB
1
receptor has been already
reported in the hippocampus of healthy dogs. Dot-like structures with CB
1
immunoreactivity
were found lining the external surface of neuronal cell bodies in the 4 regions of the CA where
the cytoplasm of neurons did not have CB
1
immunoreactivity [26]. Our findings agree with
this previous study. Interestingly, we found a progressive decrease in the immunoreactivity
from CA1 to CA4. Campora and others described a similar pattern in the canine CA [26]. In
rats [58] and humans [37] most of CB
1
immunoreactive neurons in the hippocampus are
GABAergic, and are involved in mechanism by which cannabinoids impair memory and
Cannabinoid receptor type 1 in canine nervous system
PLOS ONE | https://doi.org/10.1371/journal.pone.0181064 July 10, 2017 13 / 21
associational processes [37]. Expression of CB
1
is markedly increased specially in the stratum
pyramidale (CA1-CA3) and molecular layer of the dentate gyrus in different mouse models of
epilepsy [57,59,60] and human patients with epilepsy [60]. This CB
1
upregulation may be a
compensatory mechanism of excitatory neurons to strengthen the negative feedback loop of
the endocannabinoid system and to down-regulate neurotransmitter release [57].
The subcortical nuclei with the highest level of CB
1
receptor expression are the basal gan-
glia, including the globus pallidus and substantia nigra pars reticulate in rats [19,21], rhesus
monkeys [3] and humans [3,17]; and account for the complex effects of cannabinoids on
motor behavior [61–63]. In dogs, however, the expression is lower compared to these species
[3]. We found intense CB
1
immunoreactive fibres in the globus pallidus and in the fibres sur-
rounding unstained neuronal bodies in the substantia nigra; this immunoreactivity was stron-
ger towards the pars reticulata.
Our results show moderate CB
1
immunoreactivity in fibres and soma of neurons of the
periaqueductal grey (PAG). CB
1
immunoreactivity has been found on cell bodies [64] as well
as axons and dendrites of the PAG in healthy rats [19,64]. Furthermore, expression of CB
1
immunoreactive neurons is increased after immobilization stress [64]. Stress activates neural
systems that inhibit pain sensation depending on neural pathways projecting from cortical
neurons to the PAG and descending to the brainstem and spinal cord suppressing nociception
[64]. Moreover, antinociceptive effects of cannabinoids in the PAG have been proven in rats
[65].
Strong homogeneous staining has been described in the molecular layer of the cerebellar
cortex and surrounding the immunonegative Purkinje cells bodies in rats [19], mice [20] and
macaques [22], particularly in their basal areas, corresponding to initial axonal segments [20]
or basket cell processes [21,22]. We found identical patterns of immunoreactivity in this par-
ticular region. Higher receptor-binding levels have been found in the canine cerebellum com-
pared to humans [3], which might induce less motor depression in humans under effects of
THC [66,67]. Interestingly, the use of THC and cannabinoid analogs in experimental studies
showed ataxia and even prostration at higher dosages in dogs [67,68]. High concentrations of
cannabinoid expression in the basal ganglia and cerebellum are consistent with their involve-
ment in the initiation and coordination of movement [3,69] and explain this behavioural
changes in dogs at high doses of THC and cannabinoid analogs.
At the level of the medulla oblongata a strong dot-like CB
1
immunoreactivity was observed
only surrounding the neuronal bodies of the cochlear nucleus and the nucleus of the spinal
tract of the trigeminus. Such beaded fibers were described in the spinal trigeminal tract and
spinal trigeminal nucleus in rats [19].
Within the grey matter of all spinal cord sections, strong CB
1
immunoreactive fibres were
observed in the dorsal horn, intermediate region and ventral horn. CB
1
dot-like immunostaining
was present surrounding the body of groups of neurons. Few neurons showed slight cytoplasmic
immunoreactivity within the dorsal and ventral horns. In humans, strong CB
1
immunoreactivity
has been found in dorsal horn, lamina X and ventral horn [51]. Immunoreactive cell bodies have
been found in the lamina X in rats [23,70] and according to one study through all grey matter
[71].
DRG larger cells seamed devoid of immunoreactivity in rats [72]. Interestingly, cultured rat
DRG [73] and in-situ hybridization studies [74] showed that most CB
1
immunoreactive neu-
rons are small cells. According to our results, large neurons showed slight cytoplasmic expres-
sion and small dark neurons expressed high CB
1
immunoreactivity. Indeed, satellite cells
strongly expressed CB
1
. The presence of CB
1
receptors in the DRG and the dorsal horn may
explain some analgesic effects of cannabinoids [71]. Cannabinoids have been widely reported
Cannabinoid receptor type 1 in canine nervous system
PLOS ONE | https://doi.org/10.1371/journal.pone.0181064 July 10, 2017 14 / 21
to produce antinociception in several animal models [75–78] and the effects are mediated
through CB
1
receptors through peripheral [78], spinal [79] and supraspinal [76] mechanisms.
Moderate to strong cytoplasmic CB
1
immunoreactivity was observed in astrocytes both in
the white and grey matter along the cerebrum, cerebellum and spinal cord in all dogs. How-
ever, co-expression of CB
1
with the astrocytic marker GFAP was observed only in about ~20%
of astrocytes in the cerebral white matter. CB
1
expression has been evidenced in-situ in the
cytoplasm and processes of astrocytes in rats [43,72,80]. In vitro studies suggest that cannabi-
noids may influence astrocyte function [81]. Bidirectional neuron-astrocyte communication
has been demonstrated [82–84]. Furthermore, astrocytes have been shown to be activated by
endocannabinoids released by neurons [42]. Forming a “tripartite synapse” where an exchange
of information with the synaptic neuronal elements occurs, responding to synaptic activity
and thus regulating synaptic transmission [84]. Interestingly, increased CB
1
expression has
been shown in astrocytes of the hippocampus of epileptic rats [45]. Therefore, astrocytes
should be taken into account when assessing the overall effects of cannabinoids [44] particu-
larly in epilepsy [45].
No co-expression was found among CB
1
and the mature oligodendrocytic marker
(CNPase) in the cerebral white matter. Interestingly, previous research has shown CB
1
expres-
sion in vitro and in vivo in oligodendrocytes of healthy rat brain and spinal cord [50,85–87].
CB
1
expression was evidenced in oligodendrocyte progenitor cells (OPCs) in cultures [50,85]
and in oligodendrocytes of postnatal and adult cerebral [86] and spinal cord white matter in
rats [87]. In humans however, CB
1
receptor expression has been found in OPCs and adult oli-
godendrocytes within multiple sclerosis (MS) plaques, but not in healthy brain tissue [88].
Strikingly, CB
1
immunostaining within the sciatic nerve revealed strong expression in ran-
domly distributed Schwann cells ensheathing axons. Moreover, co-expression of CB
1
and P0, a
marker for myelinating Schwan cells was found in 100% of the Schwan cells stained. CB
1
expression in peripheral nerve fibres have been described in rats [71]. However, CB
1
expres-
sion in Schwann cells has not been previously reported to our knowledge. Nevertheless, the
presence of CB
1
in myelinating Schwann cells might have a role in myelination processes.
Regarding the age of the dogs analysed, we found a lower general CB
1
expression in the
fourth-week-old dog. CB
1
expression have been described in the fetal and neonatal human
brain showing that the density of receptor expression was generally similar in both [89] or
even higher in neonatal human brains [17]. A lower CB
1
receptor expression has been found
in aged rats in specific regions, being most prominent in the cerebellum, cerebral cortex [90],
basal ganglia [91], and less prominent in the hippocampus [90]. These findings agree with our
results and might be related to the decline of motor coordination and cognitive performance
observed in normal ageing [92].
The small number of cases of animals with different ages does not allow us to draw definite
conclusions regarding particularities among younger and older dogs. However, it is clear that
the overall distribution of CB
1
receptors is preserved in dogs at examined ages. The intensity
of the expression, however, is known to change during development and aging.
Conclusions
These results represent the first detailed spatial distribution of CB
1
receptors in the healthy
canine CNS and PNS. Our results agree with the overall distribution of CB
1
receptors reported
in other species. The high CB
1
expression found in the cerebral and cerebellar cortex, Cornu
Ammonis (CA) and dentate gyrus of the hippocampus, globus pallidus and substantia nigra
spinal cord and DRG might relate to the effects of cannabinoids on cognition, memory, motor
functions and pain sensitivity. Moreover, expression on ependymal cells and neuroglial cells
Cannabinoid receptor type 1 in canine nervous system
PLOS ONE | https://doi.org/10.1371/journal.pone.0181064 July 10, 2017 15 / 21
relate to the effects on neurogenesis and gliogenesis modulation. Finally, CB
1
expression on
myelinating Schwann cells points out potential roles of the encocannabinoid system in myeli-
nation. Our results provide a solid basis for further studies to elucidate the physiological conse-
quences and the implication of CB
1
receptors in pathological conditions with the future aim to
manipulate them in pharmacotherapy.
Acknowledgments
The authors would like to thank Petra Gru¨nig from the Department of Pathology, University
of Veterinary Medicine Hannover, Germany, for her excellent technical assistance.
Author Contributions
Conceptualization: Jessica Freundt-Revilla, Andrea Tipold.
Formal analysis: Jessica Freundt-Revilla, Kristel Kegler.
Investigation: Jessica Freundt-Revilla, Kristel Kegler.
Project administration: Andrea Tipold.
Resources: Wolfgang Baumga¨rtner, Andrea Tipold.
Supervision: Wolfgang Baumga¨rtner, Andrea Tipold.
Visualization: Jessica Freundt-Revilla, Kristel Kegler.
Writing – original draft: Jessica Freundt-Revilla.
Writing – review & editing: Kristel Kegler, Wolfgang Baumga¨rtner, Andrea Tipold.
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